TISSUE CULTURE IN FORESTRY

FORESTRY SCIENCES

Also in this series:

Prins CFL ed: Production, Marketing and Use of Finger-Jointed Sawnwood.

ISBN 90-247-2569-0
Oldeman RAA, et al. eds: Tropical Hardwood Utilization: Practice and
Prospects. 1982. ISBN 90-247-2581-X
Baas P ed: New Perspectives in Wood Anatomy, 1982. ISBN 90-247-2526-7

In preparation:

Gordon JC and Wheeler CT eds: Biological Nitrogen Fixation in Forest

Ecosystems: Foundation and Applications
Hummel FC ed: Forestry Policy
Nemeth MV: The Virus - Mycoplasma and Rickettsia Diseases of Fruit
Trees
Powers' RF and Miller HG eds: Applied Aspects of Forest Tree Nutrition
Powers RF and Miller HG eds: Basic Aspects of Forest Tree Nutrition
Rajagopal R: Information Analysis for Resource Management
Sa100 T and Madgwick HAl: Forest Biomass
Van Nao T, ECE/FAO Agriculture and Timber Division ed: Forest Fire
Prevention and Control
TISSUE CULTURE IN
FORESTRY

edited by

1.M. BONGA
Maritime Forest Research Centre, Fredericton, Canada

and

D.l. DURZAN
University of California, Davis, U.S.A.

1982
SPRINGER-SCIENCE+BUSINESS MEDIA, B.V.
 Library of Congress Cataloging_.in Publication Data
Main entry under title:
Tissue culture in forestry.
(Forestry sciences)
Includes indexes.
1. Plant tissue culture. 2. Forests and forestry.
I. Bonga, J. M. II. Durzan, D. J. III. Series.
SD403.5.T57 634.9'56 82-6292
ISBN 978-90-481-8272-5 ISBN 978-94-017-3538-4 (eBook) AACR2
DOI 10.1007/978-94-017-3538-4

Copyright 1982 by Springer Science+Business Media Dordrecht

Odginally published by Martinus Nijhoff / Dr W. Junk Publishers, The Hague. in 1982

All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or
transmitted in any form or by any means, mechanical, photocopying, recording, or otherwise,
without the prior written permission of the publishers, SpIinger-Science+Business Media, B. V.
 v

TABLE OF CONTENTS

1. INTRODUCTION 1

2. TISSUE CULTURE TECHNIQUES - J.M. Bonga 4

1. INTRODUCTION
2. LABORATORY ORGANIZATION
2.1. General layout
2.2. Facilities for tissue excision and transfer
2.3. Dish washing
2.4. Glassware and chemical storage facilities
2.5. Water purification
2.5.1. Distillation
2.5.2. Deionization
2.5.3. Reverse osmosis
2.5.4. Storage
2.6. Glassware and media sterilization
2.7. Shakers and fermentors
2.8. In~ubation facilities
3. MEDIA PREPARATION
3.1. Functions of some media components
3.1.1. Agar and its substitutes
3.1.2. Minerals, ratios, and concentrations
3.1.3. Osmoticums
3.1.4. Charcoal
3.1.5. EDTA
3.1.6. Buffers
3.2. Culture vessels and closures
3.3. Storage of nutrient media
4. PREPARATION OF CULTURES
4.1. Condition of plant material
4.2. Collection and storage
4.3. Surface sterilization
4.4. Excision and transfer of tissues
4.5. Pre-culture treatments
4.6. Incubation environment
4.7. Transfer to soil
5. CONCLUSION

3. CELL AND TISSUE CULTURE IN FOREST INDUSTRY - D.J. Durzan 36

1. INTRODUCTION
2. PRODUCTION CYCLE
3. GENETIC RESOURCES
3.1. Energy and fuel-wood species
3.2. Multiple-use species
3.3. Tropical legumes
3.4. Fiber and pulpwood species
4. PROPAGATION SYSTEMS
4.1. Seed orchards
4.2. ~ vitro vegetative propagation
VI

5. CELLS FOR COMMERCIAL PURPOSES

5.1. Creation of new hybrids
5.2. Biochemical transformation with cells and enzymes
6. CONSTRAINTS
7. OUTLOOK

4. IN VITRO PROPAGATION OF GYMNOSPERMS - A. David 72

1. INTRODUCTION
2. ORGANOGENESIS IN CALLUS AND SUSPENSION CULTURES
OF GYMNOSPERMS
3. MORPHOGENESIS IN CULTURES OF ORGANS AND ORGAN
SECTIONS
3.1. Axillary bud formation
3.2. Adventitious bud formation
3.2.1. Shoot formation on embryos and cotyledons
3.2.2. Shoot formation along the hypocotyl
3.2.3. Shoot formation on needles
3.3. Embryogenesis
3.4. Formation of shoots
3.4.1. Elongation of shoots from dormant buds
3.4.2. Elongation of shoots from adventitious
and axillary buds
3.5. Root formation
4. REGENERATION FROM EXPLANTS FROM MATURE PLANTSi
REJUVENATION
5. ESTABLISHMENT OF PROPAGULES IN SOIL
6. CONCLUSIONS

5. VEGETATIVE PROPAGATION OF DICOTYLEDONOUS TREES - 109

C.L. Brown and H.E. Sommer

1. INTRODUCTION
2. USE OF CONVENTIONAL METHODS OF VEGETATIVE
PROPAGATION IN PRODUCTION FORESTRY
2.1. Past practices and utility
2.2. Modified approaches and applications
2.3. Economic considerations using conventional
or modified propagation techniques
3. VEGETATIVE PROPAGATION VIA TISSUE AND ORGAN CULTURES
3.1. Brief historical account of organogenesis in
woody dicots
3.2. Types of cultures and their application to large
scale commercial propagation
3.2.1. Callus cultures
3.2.2. Organ cultures
3.2.3. Plantlet formation via embryogenesis in
cell suspensions
4. ECONOMIC CONSIDERATIONS
4.1. Cost comparisons of seedlings produced by tissue
culture techniques versus seedlings produced
from seed.
5. PROBLEMS ENCOUNTERED IN PROPAGATION OF TREES
USING TISSUE CULTURE TECHNIQUES
 VII

5.1. Inherent difficulties with trees

5.2. Problems associated with transplanting and
hardening off of plantlets
5.3. Production costs
6. FUTURE OUTLOOK
6.1. Use of shoot-tip cultures
6.2. Potentialities of embryogenesis

6. VEGETATIVE PROPAGATION OF EUCALYPTUS - R. Durand- 150

Cresswell, M. Boulay, and A. Franclet

1. THE GENUS EUCALYPTUS

2. MEANS OF VEGETATIVE PROPAGATION
2.1. Air layering
2.2. Grafting
2.3. Stem cuttings
3. TISSUE CULTURE
3.1. Organogenesis in callus
4. ORGAN CULTURE
4.1. Nodes
4.2. Problems encountered in developing the organ
culture technique
4.2.1. Obtaining aseptic tissue from field-grown
plants
4.2.2. Brown exudate
4.2.3. Rooting inhibitors
4.3. Factors affecting root initiation in nodes
4.3.1. Physiological state of the parent plant
4.3.2. Position on the parent plant
5. USE OF ORGAN CULTURE ON AN INDUSTRIAL BASE
5.1. Selection for cold hardiness
5.2. Introduction of clones in vitro
5.3. Multiplication of shootS-in vitro
5.4. Elongation of the shoots
5.5. Rooting of shoots
5.6. Transfer of plants to soil
6. CONCLUSION

7. VEGETATIVE PROPAGATION OF PALM TREES - J.F. Reynolds 182

1. INTRODUCTION
2. VALUE OF PALMS AND PROBLEMS ASSOCIATED WITH
THEIR DEVELOPMENT
2.1. Sources of nutrition
2.1.1. Source of edible oils
2.1.2. Source of carbohydrate
2.2. Ornamental use
2.3. Present methods of cultivation and propagation
2.3.1. Coconut palm (Cocos nucifera L.)
2.3.2. Date palm (Phoenrx-dactylifera L.)
2.3.3. Oil palm (Elaeis guineensis Jacq.)
2.3.4. Ornamental palms
3. SOLVING PROBLEMS WITH TISSUE CULTURE - CURRENT
STATUS OF RESEARCH
VIII

3.1. Cocos nucifera L.

3.2. PhOenix dactylifera L.
3.3. Elaeis guineensis Jacq.
4. PROBLEMS OF PALM TISSUE CULTURE
4.1. Obtaining explant tissue
4.2. Browning of tissue
4.3. Regeneration of adult tissues
4.4. Sterility of tissue
4.5. Regeneration frequency
4.6. Growth rates in vitro
----
4.7. Preservation of tissues
5. FUTURE RESEARCH AND PROSPECTS
5.1. Organogenesis - embryogenesis
5.2. Inflorescence reversion
5.3. Breeding programs
5.4. Disease investigations

8. PHYTOPATHOLOGY AND TISSUE CULTURE ALLIANCES - 208

H.V. Amerson and R.L. Mott

1 . INTRODUCTION
2. PATHOGEN CLASSIFICATIONS
2.1. Viruses
2.2. Bacteria
2.3. Nematodes
2.4. Insects
2.5. Fung i
2.5.1. Dual and axenic culture studies
2.5.2. Disease resistance studies with fungi
3. CONCLUSION

9. ACTION OF GROWTH REGULATORS - J.B. Zaerr and M.O. Mapes 231

1. INTRODUCTION
2. AUXINS
2 . 1. Background
2.2. Indole-3-acetic acid (IAA)
2.3. Indole-3-butyric acid (IBA)
2.4. Naphthaleneacetic acid (NAA)
2.5. 2,4-dichlorophenoxyacetic acid (2,4-D)
2.6. Other auxins
3. CYTOKININS
3.1. Background
3.2. Kinetin
3 . 3. 6-benzylaminopurine (BAP)
3.4. Other cytokinins
4. GIBBERELLINS
4.1. Background
4.2. Effects of gibberellins
5. OTHER GROWTH - REGULATING SUBSTANCES
6. CONCLUSIONS

10. NITROGEN METABOLISM AND VEGETATIVE PROPAGATION OF 256

FOREST TREES - D. J. Durzan
 IX

1. INTRODUCTION
2. IMPORTANCE OF NITROGEN METABOLISM
2.1. Range of naturally occurring nitrogenous
components in forest trees
2.2. Gene expression and mapping
2.3. Metabolic changes in organized and unorganized
systems
2.4. Nitrogen and nutrition
2.5. Aspects of intermediary nitrogen metabolism
3. NITROGEN METABOLISM IN GROWTH AND DEVELOPMENT
3.1. Precultural factors
3.2. Callus formation
3.3. Cell suspensions
3.3.1. Conifers
3.3.2. Acer
3.4. Morphogenesis
3.4.1. Nitrogen metabolism of natural embryos
3.4.2. Somatic embryogenesis
3.4.2.1. Sweetgum (Liquidambar styraciflua)
3.4.2.2. Douglar-fir and loblolly pine
3.4.3. Organogenesis
4. OUTLOOK

11. CARBOHYDRATE UTILIZATION AND METABOLISM - T.A. Thorpe 325

1. INTRODUCTION
2. NUTRITIONAL ASPECTS
3. CARBOHYDRATE UPTAKE
4. CARBOHYDRATE METABOLISM
4.1. Sucrose degradation
4.2. Metabolism of other carbon sources
4.3. Hexose mobilization and metabolism
4.3.1. Cell cycle studies
4.3.2. Growth studies
4.3.3. Organized development
4.4. Cell wall biogenesis
4.4.1. Primary cell walls
4.4.2. Cell wall turnover
4.4.3. Secondary cell walls
4.5. Carbon skeleton utilization
5. OSMOTIC ROLE
6. CONCLUDING THOUGHTS

12. THE USE OF IN VITRO TECHNIQUES FOR GENETIC 369

MODIFICATIO~FOREST TREES - E.G. Kirby

1. INTRODUCTION
2. IN VITRO SELECTION
2.1. Natural variation
2.2. Induction of variation
2.3. Selection techniques
2.4. Plant regeneration
2 . 5. Applications
x

3. SOMATIC HYBRIDIZATION
3.1. Protoplast techniques
3.2. Graft hybridization
4. GENETIC TRANSFORMATION
4.1. Principles
4.2. Procedures
4.2.1. DNA uptake
4.2.2. Transformation using biological vectors
4.2.3. Pollen as a vector in genetic transformation
5. CONCLUSIONS

13. VEGETATIVE PROPAGATION IN RELATION TO JUVENILITY, 387

MATURITY, AND REJUVENATION - J.M. Bonga

1. INTRODUCTION
2. JUVENILITY-MATURITY
2.1. Definitions
2.2. Determination in meristems
2.3. Juvenile zones
2.4. Clonal aging
2.5. Genetic stability
2.6. Mechanisms of maturation
2.7. Mechanisms of juvenility retention
2.8. Mechanisms of genetic stability
2.9. Mechanisms of rejuvenation
2.10. Sexual rejuvenation
3. SIGNIFICANCE FOR PROPAGATION BY TISSUE CULTURE
3.1. Choice of explants
3.1.1. Flower parts
3.1.2. Vegetative buds
3.1.3. Roots
3.1.4. Root-shoot junction
3.2. Chemical and physical methods of reducing
organelles
4. SUMMARY AND CONCLUSION

14. TREE SPECIES INDEX 413

15. GENERAL INDEX 416

 XI

LIST OF CONTRIBUTORS

H.V. Amerson, School of Agriculture and Life Sciences, North

Carolina State University, Department of Botany, Raleigh, N.C.
27650, USA

J.M. Bonga, Maritimes Forest Research Center, P.O. Box 4000,

Fredericton, N.B., E3B 5P7, CANADA

M. B~ulay, AFOCEL, Laboratoire de Physiologie, Domaine de

l'Etan90n, 77370 Nangis, FRANCE

C.L. Brown, School of Forest Resources, University of Georgia,

Athens, Georgia 30602, USA

A. David, Laboratoire de Physiologie Vegetale et d'Ecophysiologie

Forestiere de l'Universite de Bordeaux I, Avenue des Facultes,
33405 Talence-Cedex, FRANCE

R. Durand-Cresswell, AFOCEL, Laboratoire de Physiologie, Domaine

de l'Etan90n, 77370 Nangis, FRANCE

D.J. Durzan, Department of Pomology, 1035 Wickson Hall, Univer-

sity of California, Davis, California 95616, USA

A. Franclet, AFOCEL, Laboratoire de Physiologie, Domaine de

l'Etan90n, 77370 Nangis, FRANCE

E.G. Kirby, Department of Botany, Rutgers University, Newark,

N.J. 07102, USA

M.O. Mapes, School of Forestry, Oregon State University,

Corvallis, Oregon 97331, USA

R.L. Mott, Botany Department, North Carolina State University,

Raleigh, NC 27650, USA

J.F. Reynolds, The Upjohn Co., Experimental Agricultural Sciences

Unit 9602-25-4, Kalmazoo, Michigan 49001, USA

H.E. Sommer, School of Forest Resources, University of Georgia,

Athens, Georgia 30602, USA

T.A. Thorpe, Department of Biology, Faculty of Arts and Sciences,

University of Calgary, Calgary 44, Alberta, T2N 1N4, CANADA

J.B. Zaerr, School of Forestry, Oregon State University,

Corvallis, Oregon 97331, USA
1. INTRODUCTION

Over the past few decades tissue culture has rapidly evolved
into one of the major research tools in biology and medicine. It
has presently reached a level of sophistication where its adapta-
tion to large-scale industrial use has become possible in some
areas of agriculture, horticulture, and drug manufacturing. In
forestry, the commercial application of tissue culture is still
in its infancy, but the first inroads have been made, and further
developments can be expected.
The term "Tissue Culture" was coined in the days when the
technique was mainly restricted to the culture of pieces of tis-
sue. However, over the years the term has become somewhat of a
misnomer, because presently not only tissue pieces, but also free
cells, protoplasts, organs, and embryos are cultured.
From an experimental point of view, in vitro systems (tissues
excised from the organism and cultured in isolation) have many
advantages over in vivo ones (tissues left within the organism),
for example: 1) In the living plant the behavior of each part of
tissue is strongly influenced by correlative controls imposed by
the rest of the plant. By isolating a plant part, and culturing
it in vitro, the nature of some of these correlative controls can
be determined. 2) The isolated plant part may be free to
express potentialities that are normally suppressed in vivo, the
most obvious examples being organogenesis and embryogenesis. 3)
All in vitro experimentation is carried out under aseptic condi-
tions and therefore, the tissues and cells are not destroyed by
microorganisms. Furthermore, many chemicals can be applied over
long periods of time without these chemicals being metabolized or
degraded by microorganisms. 4) The physical environment of the
cultures is generally easy to manipulate. Most cultures are
2

grown in small containers, that fit into small incubators or

growth cabinets where temperature and light regimes are easily
and cheaply controlled. 5) In vitro culture systems are more
amenable to manipulation of the hereditary mechanisms than are
most" other systems. For example, mutants are easily induced, and
large-scale selective screening, mainly at the cellular level,
can be carried out very effectively. 6) Metabolic studies can be
carried out at the cellular rather than at more complex higher
organizational levels. 7) Factors controlling juvenility and
maturity, growth and development are often more easily studied in
vitro than elsewhere.
The potential for using tissue culture in the forest industry
is considerable, and the following applications are likely in the
next few decades; production of disease free clones, mass cloning
of selected genotypes, gene pool preservation by storage in li-
quid nitrogen, and mutant selection. Later applications may in-
clude somatic hybridization, introduction of foreign genetic
information (genetic engineering), and production of drugs and
other valuable chemical compounds. Current practical applications
are discussed in detail in this volume, particularly in chapters
3 to 8.
Since tree tissue culture is still mostly at the experimental
stage, more research is required to adapt it to large-scale in-
dustrial use. Therefore, the major current research areas, and
the theories and concepts that may determine future developments,
are reviewed in chapters 9 to 13.
This book will not deal exclusively with forest tree species.
The tissue culture problems encountered with fruit and ornamental
trees, and occasionally even non-woody plants, are often similar
to those of the major forest species and techniques developed for
the former will, after some modification, often be applicable to
culture of the latter. Therefore, tissue culture of herbaceous
and non-forest tree species will be discussed where the informa-
tion is of value for the culture of forest tree tissues. Simi-
larly, when discussing or explaining physiological control mech-
anisms, metabolism, or genetic regulation, it is often essential
to refer to studies carried out with organisms other than forest
 3

trees, because the forest tree literature simply does not provide
the required information. However, it was attempted to keep ref-
erences dealing with organisms other than trees to a minimum, and
to use literature dealing with tree species preferentially.
Tissue culture of forest trees has lagged behind that of many
agricultural crops. The main reasons for that are: 1) 'I'he long
life cycle of trees. 2) If one wishes to use mature trees, rather
than embryos or small seedlings, greenhouse material is hardly
ever available, and explants have to be taken from field grown
trees. Consequently, considerable physiological variation in
explants can be expected because of site differences and annual
fluctuations in climate. 3) Because of breeding problems, gene-
tic variation in trees is generally greater than in agricultural
crops, again resulting in variability and unpredictability in the
experiments. 4) Tissues from mature trees are often morphogenet-
ically unresponsive to the currently used experimental treat-
ments. As a consequence, obtaining in vitro veget~tive propaga-
tion is, for many tree species, still impossible or difficult. 5)
Endogenous microbial contaminants are often present, especially
in tissues of field grown material. Removal of these contaminants
is often difficult or impossible and high contamination rates are
common.
Obviously, problems still abound, and routine application of
tissue culture in forest research and industry has been lagging
as a result of it. However, as the various chapters in this book
demonstrate, the area of tree tissue culture is rapidly advanc-
ing, and new solutions for some of these problems can be expected
in the next decade or so.
4

2. TISSUE CULTURE TECHNIQUES

J.M. BONGA

1. INTRODUCTION
Tissue culture is a technique in which small tissue pieces or organs are
removed from a donor plant and cultured aseptically on a nutrient medium. By
manipulating the chemical composition of the nutrient medium and other environ-
mental parameters, the growth and development of the tissues in culture can be
directed into different channels.
Tissue culture techniques are often plagued by unknown variables. Con-
sequently results obtained in one experiment are not always reproducible in
subsequent ones, or results which can easily be duplicated in one laboratory,
sometimes are not reproducible in another. Problems may also arise when
successful routines established in small-scale initial experiments are modified
to produce the same results on a larger scale, more efficiently, and at lower
cost. Such new routines may mean slightly modified methods of media prepara-
tion, different types of culture vessels, larger growth cabinets, etc., with
each of these steps possibly introducing unsuspected unknown changes, signifi-
cantly affecting the results.
Most tissue culture techniques described in the literature are applicable
universally, although minor modifications may have to be worked out to adapt thl
technjques to local conditions. For example, in laboratories located in areas
with a warm humid climate, or in buildings with high dust levels or air drafts,
precautions to maintain asepsis may have to be much more stringent than in othel
laboratories.
Over the last few decades, there has been a steady trend towards more sophis-
ticated equipment. In a few instances this has led to easier and faster rou-
tines. For example, the weighing of chemicals, which a few decades ago was a
difficult and time consuming process is simple and fast with modern balances.
However, sophistication and automation of equipment is not always a substitute
for experience or dexterity (11, 46, 109), i.e., good results are often obtaine(
with simple, cheap equipment.
 5

For commercial enterprises with a large turnover of cultured material, rou-

tines may have to be more stringent than in a small research laboratory. In
industrial operations one would be more inclined to perform extra stringent
procedures to remove all possible sources of microbial spores from the working
area (29, 46), than in a more casually operated, small research laboratory,
because contamination of cultures means financial loss.
Many of the basic techniques have changed little since they were originally
developed, mainly by White and Gautheret. Their excellent descriptions (71,
145, 198, 199) of routines and techniques are still of great value to tree
tissue culture practitioners because much of their original work was carried out
wIth tree tissues, particularly with cambial zone explants.
With few exceptions, the tissue culture procedures for trees are similar to
those for other plants. Because plant tissue culture techniques have been
described ably and in great detail by several authors (36, 99, 166, 167), this
chapter will be restricted to some of the more general aspects of tissue cul-
ture. In particular, some aspects not always discussed in detail in the general
literature and some of the difficulties one may encounter in even the most
common and simple routine procedures will be emphasized. For the more special-
ized aspects of tree tissue culture, such as protoplast and haploid cell cul-
ture, the reader is referred to later chapters in this volume.

2. LABORATORY ORGANIZATION
2.1. General layout
Even though equipment has been modernized and some of the techniques have
changed, a modern tissue culture laboratory is still largely organized as de-
scribed by White in his classical work "The Cultivation of Animal and Plant
Cells" (198). However, if one wishes to consult more recent sources, layouts for
a modern tree propagation laboratory and greenhouse have been published (179).
Ideally, a tissue culture laboratory should have one or more sterile rooms
for tissue excision and transfer, a culture medium preparation room, separate
areas for dishwashing and chemical and glassware storage, a cold room for bulk
storage of plant material and prepared culture media, and a temperature control-
led culture room with illuminated shelves or small growth cabinets. However,
space and finances often do not allow this type of laboratory layout. In fact,
it is not uncommon to find several technicians and graduate students working in
one laboratory room without the benefit of several of the amenities mentioned
above, turning out large numbers of "clean" cultures free of contamination.
6

Therefore, simple working conditions are not always an impediment to good work.
Of course this only applies to laboratories involved in the traditional, and
simpler kinds of tree tissue culture. Those where more specialized research i~

carried out, especially in areas such as recombinant DNA and hazardous product~

will require more complex, expensive equipment and strict guidelines for opera-
tion.
2.2. Facilities for tissue excision and transfer
If the tissue culture unit is located in a building with relatively high
levels of airborne microbial spores, or if it is part of a large commercial
enterprise, proper sterile rooms may be a necessity. These are small rooms int
which air is injected through a filtering system designed to remove airborne
dust and spores. They generally have no windows (36, 168), have smooth easily
washed walls and other surfaces, and often are provided with bactericidal
ultraviolet lights to sterilize the room when not in use (36, 99, 198). SteriJ
rooms have several drawbacks; they occupy space, are expensive to build, and
most important, many staff members have misgivings about working regularly in
such a confined, featureless, and windowless environment. In many laboratorieE
therefore, sterile rooms are being replaced by laminar-flow-hoods, which are
suitable for most operations (46, 109, 168). For manipulations requiring only
small work area, a simple box without laminar-air-flow, is often sufficient
(198) (Fig. 1).
To keep sources of microbial spores and dust in the laboratory to a minimum,
petri plates, flasks, and test tubes with contaminated nutrient medium or cul-
tures should be autoclaved unopened, and cleaned as soon as possible. Sources
of microbes and small insects, such as potted plants or other plant material,
may have to be removed from the laboratory if the contamination rate of the
cultures is persistently high.
2.3. Dishwashing
Most laboratories presently have automatic dishwashers in which glassware iE
cleaned by powerful hot-water-detergent jets (46). Most of these machines rinE
in tapwater and in distilled or demineralized water to remove the detergent.
For difficult-to-clean glassware, electric ashing as a means of cleaning has
been suggested (104). For sensitive cell suspension cultures, glassware may
have to be cleaned in a chromic acid - sulfuric acid mixture. This procedure
requires strict safety precautions (168).
 7

Fig. 1. A hood for tissue excision and transfer. The hood (90 x 60 x 45 cm) is
made of wood with one slanted glass panel. In this glass panel, near the open
front of the box, are two eyepieces of a dissecting microscope (m) protruding
through a square (15 x 15 cm) hole. Around the eye pieces the hole is sealed
with a small piece of plastic film, taped to the sides of the hole. The plastic
film is flexible enough to allow up and down movement of the optics for focuss-
ing. A small alcohol flame or electric incinerator to sterilize instruments and
the mouths of glassware is placed in one of the back corners under an aluminum
heat shield (h) and ventilation hole (v). The heat shield is required to pre-
vent heat-cracks in the glass. The hood is easily sterilized by occasionally
washing its interior surfaces with 70% alcohol. Preferably, the hood is placed
on a laboratory bench in a draft-free area. To avoid excessive convection
currents inside the hood, it should not be much larger than the one shown here.
This means that the hood is suited only for routines requiring limited space.

2.4. Glassware and chemical storage facilities

Rooms for glassware and chemical storage should have abundant shelving, a
steel cabinet for flammable chemicals, and a refrigerator for heat-labile chemi-
cals. Cleaned glassware should be stored in such a manner that the inner and
outer surfaces remain free of dust and dirt. This is easily achieved by storing
the glassware in plastic bags or other containers or by covering the mouth with
a piece of aluminum foil. Preferably, chemicals should be purchased and stored
in small lots, and frequently replaced with new ones. This reduces the chances
of dust accumulation and contamination with chemicals from other bottles. Fur-
thermore, a fast turnover rate reduces the chances of hygroscopic chemicals
becoming wet and thus unstable. Most chemicals are reasonably stable as long as
they are dry and not exposed to light, particularly ultraviolet. Therefore,
most chemicals are stored in dark brown glass bottles.
8

2.5. Water purification

Although water is the most important chemical in any tissue culture medium,
often little concern is shown about its purity. However, water, even if it is
relatively pure, may by its sheer volume contribute more impurities to the
culture medium than the glassware, instruments, agar, or nutrient chemicals .
Pure water is somewhat of a myth. Terms such as "triple distilled", "ultra-
pure", etc. can be misleading, because such claims often are based solely on
electrical resistance measurements that measure ion concentration, but not the
many non-ionized impurities that may be present in the water (6, 74, 97). Fur-
thermore, even if good water is produced, it will deteriorate rapidly if collec-
ted improperly or if stored for any length of time.
Most tap water contains minerals, silt, oils, metallic oxides, pipe corrosio l
products, organics, microorganisms, and dissolved gases (140). The most common
methods to rid water of such impurities are distillation, ion exchange, and re-
verse osmosis, sometimes performed singly, or more commonly in various combina-
tions.
2.5.1. Distillation. Distillation has traditionally been the major system
for water purification, and where water is required only in small quantities, i
is still generally the preferred method to produce good quality water. Proper
distillation is a technically complex procedure. In stills that are not proper-
ly designed or operated, many impurities may be transferred to the receiving
vessel, either by mist or film flow, or in the case of volatiles with a boiling
point close to that of water, by distillation (6, 73, 85). Stills do not elimi-
nate all minerals, but they effectively remove most large organic molecules,
including the highly toxic pyrogens. To remove smaller, generally more volatil.
organic molecules, potassium permanganate or other oxidizers are sometimes adde.
to the boiler to degrade these organics to carbon dioxide and water. However,
many of these organics are not immediately broken down to carbon dioxide and
water, but to low molecular weight intermediates. These are often more volatil.
and thus more likely to be distilled with the water than are the original mole-
cules, thus increasing the amount of impurities in the distillate (72).
Ammonia, low molecular weight aliphatic acids, chlorine, and some amines are
difficult to remove by distillation (43, 73, 85). In fact, in some stills thes.
chemicals will accumulate in concentrations in the distillate that are higher
than their concentration in the feeding water. For that reason, if improper
equipment is used, double or triple distilling may result in accumulation of
some impurities in increasing concentration in each successive distillation.
 9

To obtain satisfactory results with a still, the following rules, should be

adhered to (72, 73): 1) The condenser should be hot enough to allow venting of
some of the volatiles carried in the steam. For most stills, the cooling water,
at point of entry, should be about 10C (73). 2) Discard the water produced
during the first 10-15 minutes of still operation. During the warm-up period
some low boiling point volatiles are distilled before steam is formed, and some
of these volatiles may accumulate in the water collected during the first few
minutes at concentrations up to hundreds of times their concentration in the
feed water (72, 73). 3) The still boiler should be drained and cleaned after
every few hours of operation to remove accumulated impurities.
2.5.2. Deionization. As water passes through an ion exchange column, ionic
impurities are removed. This process produces water virtually void of all ionic
material. Because stills are only partially effective in removing ions from wa-
ter, ion exchange columns are often used to produce the feed water for a still.
Ion exchange columns should not be used as the only means of water purification,
or after other methods of purification, because ion exchange columns often re-
lease large quantities of organic contaminants. These include phthalate ester
plasticisers, plastic auto-oxidation products, and non-ionized nitrogenous
compounds leaching from the resins (43, 72, 99). Furthermore, microorganisms
generally thrive well on the resin beads, producing a large array of toxic and
non-toxic organics (72). Therefore, even though the demineralizer will remove
ionic organics from the water, it will add many others. However, most of these
will be removed by distillation, if the demineralized water is fed into the
boiler of a well functioning still.
Another problem with demineralizers is that small resin particles are often
dislodged from the bed and enter the water stream. If these enter the boiler of
a still, they are broken down to soluble organics, which may be only partly
removed by distillation (72). This problem can be prevented by placing a cellu-
lose or other filter in the water line between the demineralizer and still. A
distinct advantage of having a demineralizer feeding a still is that it prevents
the formation of scale in the boiler of the still.
2.5.3. Reverse Osmosis. Water purification by reverse osmosis has gained
popularity lately. It is especially suited for institutions requiring large
quantities of purified water. The reverse osmosis membrane or cartridge will
eliminate microorganisms, particulate matter, and molecules with a molecular
weight greater than about 300, including pyrogens (68, 72, 97). For further
10

purification, a demineralizer or a still may be added. The demineralizer should

be small and of high quality to avoid, as much as possible, the reintroduction
of resin leachates, and microorganisms and their breakdown products into the
water (72). In some systems, the demineralizer is followed by a membrane filter
with 0.22 ~m por e s to remove microorganisms and particulate matter.
2.5.4. Storage. The length of time water is kept in storage should be as
short as is practically possible. Stored water will leach various chemicals
from plastic or glass containers and rubber or other tubing (14, 72, 85, 105,
149), and, if in contact with the atmosphere, will accumulate vol a tiles, dust,
and microorganisms (72). Some bacteria grow rapidly in stored distilled water,
reaching high population density levels (61).
2.6. Glassware and media sterilization
There are several means of sterilizing glassware and culture media. These
include sterilization by radiation, ethylene oxide, dry heat, autoclaving, sol-
vents, and filtration (16, 21, 34, 93, 168, 186). Sterilization by radiation or
gaseous ethylene oxide are not often used in plant tissue culture, and will not
be discussed further. Dry heat sterilization is restricted to glassware and
some instruments, and is carried out in an oven at 140-160C for several hours
(186, 198). The most common method for sterilizing glassware and nutrient media
is autoclaving, generally at about 120C for 15 minutes. In electrically or ga~

heated autoclaves, it is advisable to use demineralized water to feed the boil-

er. Especially in areas with a hard-water supply, the water level and pressure
control valves tend to become rapidly clogged with scale and will cease to func-
tion properly if the water is not deionized before entering the autoclave. SomE
autoclaves are run on centrally supplied steam. Such steam is often contamina-
ted with high levels of various volatiles, some of which may be absorbed by the
glassware and nutrient media in the autoclave (9). Contamination of glassware
and nutrient media by volatiles may also occur if the nutrient vessels are wra~

ped in paper before autoclaving, the volatiles being generated from the paper b)
the hi gh temperature steam (9, 198). Another problem can be the formation of
volatile inhibitors from rubber stoppers and tubin g during autoclaving (22).
The media should not be autoclaved in large volume in one vessel, but in
small volumes in several vessels. The larger the volume of the medium, the
lower the surface to volume ratio, and the poorer the heat exchange. For exam-
ple, several litres of medium in one flask will not reach the maximum tempera-
ture of the autoclave if autoclaved for the usual 15 min. If autoclaved longer ,
to reach hi g her temperatures, there is the danger of violent boiling during
 11

cooling of the autoclave, because of too rapid a drop in pressure.

Lately, a new type of sterilizer has appeared on the market. In this steri-
lizer ("Agarmatic", N.B.S. Co.) (Fig. 2), the nutrient chemicals, including the
agar and the required amount of water, are added directly to a 3-litre stainless
steel pressure vessel. The pressure vessel has a stirrer, which effectively
dissolves the chemicals in the water during the heating and sterilizing cycle.
Heat exchange is very efficient in a continuously stirred solution. This assur-
es a fast heat-up before, and rapid cooling after sterilization, which keeps
thermal breakdown of the chemicals to a minimum (122). After cooling, cold-
sterilized heat-labile chemicals can easily be added to the stirring nutrient
through a porthole in the lid of the pressure vessel. To dispense agar contain-
ing nutrients, the dispensing temperature of the sterilizer is maintained at
about 60C.

Fig. 2. A bench top sterilizer with a propeller (p) to keep chemicals in solu-
tion during autoclaving, and with an entry port (e) to add filter-sterilized
chemicals. A dispensing pump (d) is used to transfer the nutrient to the cul-
ture vessels.

Many chemicals will partially decompose when autoclaved. For example, carbo-
hydrates, particularly at a slightly acid pH, will undergo some degree of hy-
drolysis and further breakdown when autoclaved (13, 17, 122, 130, 141, 142,
168). Fructose will produce small amounts of toxic furfurals in normal auto-
claving (122, 130, 141). Sugar decomposition is stimulated if the sucrose is
autoclaved together with iron and phosphate ions (170), and sugars interact with
12

amino acids when heated together (122, 130). Most vitamins (82) and gibberellic
acid (28, 139) are heat- labile, but the commonly used auxins (except indoleace-
tic acid) and cytokinins are relatively stable (53, 136).
However, even though autoclaving induces chemical changes in the nutrient
medium, it is still the preferred method of sterilization, except for a few very
heat sensitive chemicals. The main reasons for this preference are: 1) The
operation is simple and effective. 2) As long as the duration of autoc1aving is
not extended past the usual 15 or 20 minutes at about 120C, the chemical chang-
es are small and generally have little or no noticeable effect on growth of the
cultures (202). 3) The autoclaving effect is not always a neutral or negative
one;-for example, Ball (13) found better growth of Sequoia sempervirens callus
on a medium with autoclaved sucrose than with filter sterilized sucrose. In
some media, inhibitors are inactivated by autoclaving (122).
Heat-labile chemicals, such as glutamine and some of the vitamins, are
cold-sterilized and added to the autoclaved portion of the medium (70, 194).
Cold-sterilization is sometimes carried out by dissolving the chemical in a
small amount of solvent, generally dimethylsulfoxide (91) or ethanol (53, 125,
135). However, ethanol may not be a good choice for this purpose. Concentra-
tions of 1% (135) and lower (53, 125) in the medium will inhibit callus growth
and have been found to inhibit embryogenesis (181). The more common method of
cold-sterilization is by filtration through membrane filters. Filtration tech-
niques have evolved rapidly over the last few decades and have found many indus-
trial and laboratory applications. As a consequence, a large variety of fil-
ters, primarily membrane filters, are now commercially available to remove
microorganisms from solutions. Most filters are made of cellulose acetate and
have 0.20 ~m pores. The most popular method of filtration is vacuum filtra-
tion in which the solution is placed in a filter funnel and sucked through the
membrane into a vacuum flask. Before use, the filter, its funnel, and the vacu-
um flask are sterilized by autoclaving or with alcohol (27, 106), the latter
method being quicker and more convenient. Vacuum filtration has a few disadvan-
tages. If a water run vacuum aspirator is used, irregularities in the water
flow may cause a backflow of air or water into the vacuum flask, introducing
contaminants to the filtrate. Furthermore, if the filtrate contains organics it
may foam in vacuum and some of the more volatile organics may partly be removed
by evaporation. To avoid these complications, it is probably better to use a
pressure rather than a vacuum filtration system (Fig. 3).
There are some problems associated with membrane filters. Often these fil-
 13

ters contain a small amount of detergent, some of which is released into the
filtrate. Most cell cultures will not be affected by trace amounts of detergent
in the nutrient, but the possibility that the growth of some sensitive cell
populations could be influenced cannot be ruled out. Rinsing of the membrane
filter in water will remove the detergent, but reduces filtration speed (34,
38). Adsorption of proteins and some other media components to the filter and
oxidation of sensitive chemicals may occur (84, 122). Furthermore, 0.20 ~m

polycarbonate filters may pass some bacterial species; cellulose ester filters
of the same pore size will remove these bacteria (131).

Fig. 3. Various membrane filter systems are used for cold-sterilization of

chemicals; vacuum (v), air pressure (a), and the simplest and most practical
system, syringe pressure (s).

2.7. Shakers and fermentors

It is generally easier to maintain tissues or obtain vegetative propagation
on a semi-solid agar medium or on a filterpaper wick in a liquid medium than in
agitated liquid medium. However, agitated liquid cultures potentially have
several advantages: 1) Agar, which is chemically complex and variable (199), is
deleted, thus assuring more uniformity in culture conditions. 2) In those cases
where vegetative propagation is possible in agitated liquid cultures, often far
more propagules are produced than in stationary cultures. 3) Agitated liquid
cultures are more suitable for biochemical research (60) and studies involving
synchronization of cell division (75) than are stationary ones. It is expected,
therefore, that liquid shake cultures will eventually find wider application in
14

tree tissue cultures than at present.

The most common means of agitation of liquid media is by placing the culture
vessels on a drum (for test tubes) or a disk (for flasks) slowly rotating around
a near horizontal axis (169). This assures good aeration of the liquid cultures
with little mechanical damage to the cells. Agitation can also be achieved by
placing culture flasks on reciprocating or gyrotory platform shakers. Gyrotory
(horizontal rotary orbit) shakers are preferred over reciprocating (horizontal
linear motion) ones for a variety of reasons (66). The performance of the
gyro tory shaker is determined by the speed of agitation and angle position of
the flasks, with optimal aeration occurring in flasks tilted about 45 from the
vertical (66). A new shaker design is the "tapping" motion shaker in which the
culture is agitated by a magnetic bar moving up and down. This vertical move-
ment of the bar is less injurious to cells than the rotary movement of the bar
at the bottom of the flask used in some conventional culture systems (92).
For large-scale and continuous (steady state) cell culture, fermentor type
systems ("chemostats") are often used. In most of these the gaseous environment
is controlled by the bubbling of air or mixtures of various gasses through the
nutrient, and part of the nutrient is regularly withdrawn and replenished. Such
systems have been described in detail (169, 171) and have been used extensively
for metabolic studies of tree tissu~s, e.g. of Acer pseudoplatanus (60, 65,
171). If gasses from pressure tanks are bubbled through the nutrient, only high
quality gasses should be used. Air and gas pressure tanks often contain appre-
ciable amounts of ethylene and other impurities that could affect the cells in
culture (58).
2.8. Incubation facilities
If only a few cultures are to be incubated, controlled environment cabinets
can be used. However, if more space is required, controlled temperature rooms
are used. These often cannot be programmed for variable temperatures, and
therefore, are run mostly at one constant temperature, or at a constant day and
a lower constant night temperature. To expose all cultures to more or less
equal light intensi ties, the cuI ture vessels are generally placed on tiers of
shelves, with each tier having its own banks of fluorescent lights. Generally,
only low intensities (1000 lux or less) of light are used, either constantly or
at specific photoperiods. Overheating of the atmosphere in the vicinity of the
lights can be a problem.
If culture vessels are used that are not hermetically sealed (with screw
caps) but are covered with closures that allow some air exchange, the cultures
 15

should be protected from air currents. Slow growing cultures can be placed in-
side clear polyethylene bags; for faster growing cultures the bags may have to
be punctured to allow a more rapid air exchange. Polyethylene is an excellent
barrier to water vapor, but allows exchange of carbon dioxide and a less rapid
exchange of other atmospheric gasses (151).
Volatiles in the culture room atmosphere may create some problems for extra
sensitive cultures, especially if ventilation of the rooms is restricted. There
are various sources of volatiles; air conditioning units may leak refrigerant
(freon or ammonia), fluorescent lights produce ethylene (200), and gasses
emanate from electric motors and insulation material (33). However, normally
these volatiles will not reach levels high enough to have a significant effect
on the cultures.
One problem often encountered in culture rooms is contamination of cultures
by fungi carried by mites (Ill). Placing the cultures in tightly closed poly-
ethylene bags does not exclude the mites. Presumably they are attracted by
volatiles, produced by the cultures, that pass through the plastic of the bag.
The mites appear to be capable of drilling through the plastic and crawling into
the culture tubes carrying fungi with them. To rid the culture room of mites,
bench tops and shelves should be washed with 70% alcohol and floors and walls
wi th a sodium hypochlori te cleaner. Hanging a few "Vapona" (d ichlorvos, Shell)
insecticide strips in the culture room for a few weeks will further eliminate
the insects without noticeably affecting the cultures. Several other miticides
of low phytotoxicity are available (Ill, 143, 162).

3. MEDIA PREPARATION
Tree tissue cultures have been maintained on a large variety of different
nutrient media. The chemical comp0sition of these is not discussed in detail
here, because several of the later chapters in this volume will deal at length
with the currently used nutrient formulas, the functions of nitrogen, carbohy-
drates, and hormones in the media, and other aspects of nutrition. Complete
nutrient formulas can also be found in textbooks on plant tissue culture (71,
166, 170) and reviews of nutrient media (70, 86, 145). Therefore, this discus-
sion will focus mainly on a few principles and problems not always emphasized in
the general literature.
3.1. Functions of some media components
The primary function of most components of the medium is nutritional, i.e.,
they provide energy or serve as building blocks for other essential molecules in
16

the plant cells. However, some components have functions that are mostly non-
nutritional, and sometimes more physical than chemical in nature. These non-
nutritional functions have received less attention in the literature than the
nutritional ones, and therefore will be emphasized in this section.
3.1.1. Agar and its substitutes. As was pointed out earlier, liquid suspen-
sion cultures are often preferred over semi-solid agar cultures. However, for
many tissues, techniques for cell suspension cultures have not yet been perfec-
ted and the tissues still survive or grow better (50, 117, 174), or, as is the
case in cultures of Eucalyptus (52), are more morphogenetically active, if cul-
tured on an agar medium. The reasons are not clear, but the following may be
suspected: 1) Loss of vital chemicals from the cells by leaching may be more
severe in liquid medium. 2) Agar, besides providing a solid support for the
tissues, could be beneficial because it has an adsorptive capacity (199), and
like charcoal, may remove some cellular waste products. 3) Cells in agitated
liquid medium are prone to mechanical damage.
One advantage of cultures on semi-solid agar media over liquid suspension
cultures is that they do not require expensive and bulky shakers. On the other
hand, because agar is a natural product, one may expect differences in growth
response of the cultures, depending on the degree of purification of the agar
(146), and with different batches. Furthermore, because of dwindling supplies,
good quality agar is sometimes difficult to obtain and is becoming expensive,
although the cost can be reduced by recycling agar from old cultures (10).
Another problem with agar is that it is a source of many minerals, in particular
sodium (89, 132) and possibly some vitamins (132, 199, 202) and toxins (102,
132), which may complicate metabolic and nutritional studies.
Therefore, several agar substitutes have been investigated, but so far, none
have been widely accepted. The most interesting of recently developed substi-
tutes are; positively charged dextran microspheres (107), "Plantgar", a starch
co-polymer (44), polyac rylamide (86, 119), silica (144) and "Ficoll", a sucrose
polymer (184). Instead of gelling agents, glass beads (47), filterpaper, glass
fiber or polyester (40, 86) are sometimes used to support the tissues. Glass
fiber supports should be washed in acid to remove chemical contaminants before
they are used (176). When using agar, its concentration is important; morpho-
genesis as well as callus growth rates are influenced by its concentration (102,
146,184,199).
3.1.2. Minerals, ratios, and concentations. If one looks at the macro-ele-
ment composition of different nutrient media one will notice large differences
 17

in the concentrations of various salts and in total salt concentration. Both

aspects have a considerable effect on growth rates and morphogenetic patterns.
For example, short exposure of tissues to a medium rich in calcium and nitrate
but without potassium stimulates rooting in some cultures (185). High potassium
is sometimes required for embryogenesis (32), and media high in phosphate (117)
or low in ammonium nitrate (133) are used occasionally to induce shoot forma-
tion. In other situations the concentration of each element and the ratios
between various elements, though important, may be less influential than the
concentration of all salts combined. For example, callus cultures of Pinus
coulterei showed optimal bud formation on a 1.5X normal strength salt solution,
while in cultures of i. taeda the optimum occurred at the much lower concentra-
tion of 0.5X normal strength (20); optimal bud formation in Norway spruce and
Douglas fir cultures occurred at 0.5X, or lower strength of the nutrient solu-
tions (39, 87). Furthermore, rooting often depends on a low total salt concen-
tration (133, 134).
3.1.3. Osmoticums. Sucrose is the main mobile carbohydrate in plants and is
the carbohydrate most commonly used in tissue culture as energy source and as
osmoticum (65). Many cultures, especially embryo cultures, perform properly
only when cultured on a nutrient medium with a high osmotic potential, and for
shoot formation, a higher osmotic potential is sometimes required than for
callus growth. Generally, the osmotic potential of the medium is controlled
with sucrose, but other osmotic agents can be used. If enough sucrose is pro-
vided to satisfy the energy needs of the tissue, the remainder of the osmotic
requirement can be provided by a non-metabolizable sugar (30, 31). Mannitol is
generally used for that purpose, although instances are known where mannitol is
metabolized by the tissues, e.g., in Fraxinus callus cultures (202). High
sucrose concentrations promote maturation and senescence (81), which is often an
undesired effect. In Citrus, omission of sucrose in one subculture, or replac-
ing sucrose by the less easily metabolized sugars, galactose and lactose, stimu-
lates embryogenesis (100, 101). However, galactose is toxic to many plants
(54), and thus may not be universally suitable as an osmoticum or metabolite to
induce morphogenesis. In pear cultures, cell volume, growth rates, and mortal-
ity were controlled by mineral and mannitol concentrations (45). Sometimes pol-
yethylene glycol is used as osmoticum. Preferably, it should have a molecular
weight higher than 1000, because lower weight molecules penetrate the cells
(126).
3.1.4. Charcoal. Activated charcoal is added to nutrient media mostly to
18

remove toxins present in agar (102), or aromatic waste products excreted by

cultured tissues (67, 191). It adsorbs aromatic molecules preferentially over
straight chain ones, and the larger the molecule the stronger the adsorption
(5). Charcoal prevents browning of tissues (180) and stimulates embryogenesis
and rooting (67), although reduced rooting has also been reported (147). The
possibility that some of the stimulating effect on rooting, could, in part, be
the result of the charcoal having some soil-like properties (exclusion of light.
and possibly some ion exchange capacity) should not be overlooked (59, 138).
In the tissue culture literature it is often not specified what kind of acti-
vated charcoal is used. However, such specification is important, because con-
siderable differences in adsorptive characteristics exist depending on how it il
manufactured (5). Charcoal of plant origin is different from charcoal of animal
origin. Wood charcoal contains up to 98% carbon, bone charcoal, unless acid
extracted, often contains only about 10%, the rest mainly being calcium and
phosphate (148). Bone charcoal also contains some drug-like compounds and
cyanide (183), which, if the charcoal is used in high concentrations, could
affect the metabolism of some cultures.
3.1.5. EDTA. Ethylenediaminetetraacetic acid (EDTA) and related chelates
are added to the medium to keep iron in solution. However, besides chelating
iron and other minerals, EDTA has several side effects that are worth noting.
For example, in a few cases iron chelates stimulated embryogenesis and root
growth, where iron dissolved in non-chelated form did not (153, 164). EDTA,
without iron, at concentrations comparable to those used in tissue culture
media, stimulates nitrate reductase (115) and inhibits ethylene formation (155)
which may be significant because reduced nitrogen stimulates embryogenesis
(197), while ethylene inhibits it (182). Pretreatment of shoots of mature
conifers with EDTA without iron, stimulated morphogenesis in these shoots after
their transfer to culture medium (26).
It has recently been pointed out that in several widely used nutrient media,
EDTA is not equimolar with iron, but is present in excess amounts (159).
3.1.6. Buffers. The pH of culture media tends to shift, especially in
liquid media (55, 120, 197). It is difficult to correct this problem with
buffers because buffers are only effective if they are non-toxic and are not
removed from the medium by the cells. Buffers in that category are 2-(N-morpho-
lino)ethane sulfonic acid (MES) and Tris(hydroxymethyl)methylamine (TRIS) (127,
128), although in some tissues TRIS strongly affects several physiological pro-
cesses (187), which may restrict its use in tissue culture. Another method of
 19

keeping the pH more or less under control is by frequent transfers to fresh

media, or in liquid cultures by frequent pH adjustments.
3.2. Culture vessels and closures
For semi-solid cultures, petri dishes, erlemeyer flasks, bottles, or test
tubes are used. Petri dishes and flasks have the advantage that the nutrient
has a large surface to volume ratio, which ensures good aeration of the nutrient
medium. Petri dishes have the disadvantage of easily becoming desiccated and
contaminated. Test tubes have the advantage that they are cheap, many fit in a
small space, and desiccation and contamination rates of nutrient media in test
tubes are generally low. A disadvantage is that the nutrient surface area is
small, and, therefore, nutrient aeration is limited. Consequently, test tubes
are suitable only for relatively slow growing cultures.
For liquid cultures, flasks, test tubes, or specialized vessels (169) are
used. In most of these, aeration of the nutrient is largely determined by the
method of agitation of the nutrient (section 2.7.).
The volume of both atmosphere (56) and nutrient (86) in the vessel will
affect growth rates and morphogenesis and thus should be standardized. Similar-
ly, proper ventilation of the atmosphere in the vessels is important because
accumulation of volatiles may inhibit morphogenesis (42, 181) and other process-
es (49). Rapid changes in atmosphere, for example, when vessels are opened, may
result in temporary "gas-shock" of the cultures (178).
The most common vessel closures are polypropylene or stainless steel caps and
foam plugs, each allowing some air exchange while providing protection against
contamination and excessive desiccation of the nutrient. A recent development
is a polypropylene cap (Kimble) with a membrane permeable to most atmospheric
gasses but not to water vapor. Differences in growth rates were observed in
vessels closed with polyethylene and aluminum foil (49).
3.3. Storage of nutrient media
In a survey by the "Tissue Culture Association" (80) concerning storage of
culture media for animal cell cultures the consensus was that most media could
be stored refrigerated in liquid form for several months and sometimes for more
than a year without this resulting in reduced growth rates. In some media,
precipitates appeared after a few weeks in storage. In one medium, a 50% loss
of leucine and glutamine was noted over a 4-month storage period. Media in
powder form stored at 5C for two years showed no loss in growth-promoting
capacity.
Loss of moisture during storage is a problem (122). Keeping the vessels with
20

nutrient media enclosed in plastic bags will reduce desiccation and keep out
dust and contaminants. Slow chemical changes will occur in refrigerated cultur,
media (122). L-glutamine in liquid medium is largely destroyed in about four
weeks (7). Some stock solutions for plant tissue culture media can be stored
froze~ (70). A few chemicals are highly unstable in liquid solutions. For
example, ascorbic acid was lost at a rate of 4% per day from nutrient media
stored at DoC, while at room temperature its half life was only 15.5 h (63).
Indole acetic acid decomposes in solution, especially if exposed to light (18,
23).

4. PREPARATION OF CULTURES
4.1. Condition of Plant Material
In tree tissue cultures results often vary greatly from year to year, even
when the ex plants are obtained from material collected from the same tree at th,
same time each year. This probably is partly caused by climatic cycles and
fluctuations in other environmental factors. For example, annual growth in
conifers is affected by spring rainfall, and annual nitrogen uptake by rainfall
in June; both these processes are correlated with climatic cycles lasting from
4.4 to 42 years (116). Such cycles in the physiological conditions of field
grown trees could account for some of the year-to-year variation in culture
response. A very short cycle has been observed in rooting response of genet-
ically uniform Pinus radiata cuttings; however, which environmental component
controls this cycle is not known (161). In conifer cultures the rate of adven-
titious bud induction on cotyledons is correlated with the growth rate of the
parent tree (123) and with seed size (3), both of which are largely determined
by environmental factors.
In seedlings or small sized plants, variation in the physiological condition
of the plants can be reduced by raising them in controlled environments, i.e.,
in greenhouses or growth chambers, instead of in the field. The morphogenetic
capability of greenhouse raised plants is improved by adherence to optimal
light, temperature, and fertilization schedules (64). However, if the objectiv(
is to culture tissues from mature trees, greenhouse material is only rarely
available, and field grown material will have to be used. In such material,
environmentally induced variations in the physiological condition are unavoid-
able.
4.2. Collection and storage
In the early days of tree tissue culture, most experiments were carried out
 21

with callus cultures from cambial zone explants. White's (198) and Gautheret's
(71) textbooks and some other early references (76, 199) give extensive descrip-
tions of the collection and subsequent handling of material for the preparation
of cambial zone cultures. However, over the years the interest in cambial zone
cultures has waned, because these, though providing good callus growth, often
have a low morphogenetic capability. Therefore, interest has shifted to cul-
tures of embryo or young seedling sections, shoot tips, buds, microsporophylls,
anthers, pollen, and female gametophytes.
Good quality seeds to provide embryos, female gametophytes, or small seed-
lings can be obtained commercially or are often available from government for-
estry institutions. Dry seeds of most trees can be stored in sealed containers
at 3 to -18C for several years without loss of viability (190) .
Branches with vegetative buds or generative structures, to provide shoot
tips, young foliage, microsporophylls, anthers, or pollen, are collected in the
field and transported to the laboratory in plastic bags. One problem is that
microsporophylls, anthers, and, in some cases, vegetative shoots are suitable
for culture only if excised from material collected durin g specific short
periods each year. For example, balsam fir shoots are morphogenetic only if
excised from buds collected shortly before the spring bud break, i.e., during
the short period when the lateral meristems are formed inside the bud (26), or
if excised from late summer or early fall buds, i.e., during the peri od when the
needle primordia are formed (Bonga, unpublished) .
Therefore, if one intends to use such material on a year-round basis, proper
storage methods are imperative. Conifer branches will remain viable for several
months if stored at 4C in paper bags placed inside plastic bags (Bonga, unpub-
lished). The function of the paper bags is to absorb excess surface moisture
from the branches, thus reducing fungal contamination. Extensive studies have
been carried out with regard to fruit and vegetable storage, involving gaseous
mixtures (35), reduced air pressure (108), fungicides (83), specific relative
humidities (19), and growth regulator treatment (79). Some of these methods
have been used for storing tree seedlings (189). For cold storage of tree seed-
lings it has been recommended to use Kraft paper bags with a semipermeable
polyethylene lining, which restricts moisture loss but allows sufficient gas
exchange to maintain the seedlings in a healthy state (113, 189). To reduce
moulding, the seedlings are sometimes wrapped in damp sphagnum before being
placed inside the storage bags (189). Apple shoots have been stored in vitro at
low temperature for 12 months (110). Storage of normally short-lived citrus
22

pollen was improved by using a nitrogen atmosphere and low temperatures (152).
When material is stored at low temperature under conditions of restricted gas
exchange or altered atmospheric conditions, slow, degenerative physiological
changes occur. Pine seedlings lost carbohydrate in cold storage (112, 189),
while in other plants, alcohol, acetaldehyde, and abscisic acid levels increas-
ed, and chloroplasts and mitochondria were inactivated (41). Therefore, one can
expect explants from stored material to behave differently in culture than
explants from fresh material.
Cold storage is not always detrimental. In some cases it is required to
break dormancy, while in others, it has a beneficial effect in non-dormant
material. For example, 6 to 8 weeks of cold storage of non-dormant branches of
balsam fir before excision and culture of young shoots stimulated the formation
of adventitious structures on the expanding needles (26).
A technique that is presently being developed, mainly for germplasm storage,
is cryopreservation, i.e., storage of tissues in liquid air or liquid nitrogen.
With tree tissues, such storage has been achieved for poplar callus (154) and
suspension cultured sycamore cells (172).
4.3. Surface Sterilization
It is often difficult to surface sterilize material without simultaneously
damaging or killing the tissues. In fact, when extra sensitive tissues, such as
shoot apical meristems, are to be excised it is sometimes better not to use any
sterilant (146). Sometimes microorganisms are endogenous, e.g., in seeds (173),
in dormant winter buds but not in the spring flush of pecan trees (98), and on
the bud scales but not the leaf primordia of apple trees (4). Tree tissue cul-
tures free of such microbes can often only be obtained if the explant is limited
to the shoot apical meristem (24).
The most common surface sterilants are calcium and sodium hypochlorite, with
the calcium salt being less toxic to tissues than the sodium salt (173). How-
ever, calcium hypochlorite reacts with carbon dioxide in the atmosphere, and,
therefore, is chemically unstable (156). Hypochlorite has a negligible activity
at pH over 8 (17); buffered at about pH 6 it is much more effective (173).
Generally a drop of detergent is added. However, in a surface sterilization
experiment with white spruce twigs it was found that adding detergent, though
lowering the contamination rate, increased the toxicity of hypochlorite to the
explants (25). A short exposure to hypochlorite followed by a 2-min wash in 70%
ethanol has been effective for conifer buds and shoots (12, 26). All hypochlor-
ite should be removed from the tissues, because trace amounts left behind will
 23

interfere with amino acid uptake and metabolism. Washing several times in water
does not always remove it sufficiently; washing in dilute hydrochloric acid may
be required (1, 2).
Mercuric chloride in 50% alcohol has been used to surface sterilize pine
brachyblasts (51), and in water, has been used to treat shoots of sandalwood
(160). If used to sterilize conifer seeds, mercuric chloride stimulates imbibi-
tion and consequently germination. However, mercury ions are adsorbed to the
seedcoat and have to be removed by washing in potassium chloride (177).
Alcohol without other sterilants has been used to surface sterilize pine
seedlings (188) and poplar shoots (37), but generally alcohol is used in combi-
nation with other chemicals. The effectiveness of alcohol depends on chain
length of the molecule and the presence or absence of water (137).
Hydrogen peroxide has been used to surface sterilize shoots of saltbush (201)
and poplar (37). For stem sections of some conifer species, it was more satis-
factory than sodium hypochlorite (76). Various simple acids and bases were
effective in surface sterilizing carrot roots (157); merthiolate was more effec-
tive than sodium hypochlorite and hydrogen peroxide for green alder seeds (88).
4.4. Excision and transfer of tissues
When tissues are cut, the cut surfaces often turn brown within a few minutes
because of oxidation of phenols to toxic quinones (118) in the damaged cells.
The smaller the explant, the higher the cut surface to volume ratio, and thus
the higher the degree of damage. This probably is one reason why it is often
difficult to achieve survival of small explants. To keep cell damage (crushing)
to a minimum the scalpels used for excision should be as sharp as possible.
Therefore, to sterilize them, they should not be heated unnecessarily, because
overheating dulls the edge. The common procedure is to dip the scalpel in
alcohol, which is then removed by igniting it. The combination of the steriliz-
ing effect of the alcohol and the mild heat created when it is burned off is
enough to sterilize the scalpel without damaging its sharp edge (175).
Additional treatments are often required to prevent browning. Excised tis-
sues of coffee (118), palm (180), and balsam fir (26) were treated with various
antioxidants, tissues of peach with the herbicide "Dieca" (121), and those of
teak and jojoba with polyvinyl-pyrrolidone (78, 150). Keeping tissues submerged
in water during excision of the explants results in less browning and improved
survival rates of the cultures, presumably by excluding air from the cut sur-
faces (26).
Browning is not the only problem associated with excision. There is a short
24

period of rapid loss of adenine nucleotides and potassium from freshly excised
tissues, which can be counteracted by washing in calcium chloride (77), and of
intercellular carbon dioxide (103). Various types of stress caused by injury,
and recovery from these stresses have been described (69, 103). Whenever excis-
ing tissues, these stresses should, if possible, be identified and counteracted
as much as possible. In addition, for very delicate and small explants, special
tools and excision methods may be required to keep cell damage to a minimum (48,
175). However not all wounding effects are negative, e.g., in cultures of
jojoba (150) and apple (165), rooting was induced by wounding.
After excision and transfer of the explants to the culture vessel it is
customary to flame the mouth of the vessel in a small alcohol or gas flame
before recapping the vessel. However, it has been found that this procedure
introduces a significant amount of ethylene into the culture vessels (15).
Therefore, if tissues that are extra sensitive to ethylene are to be excised and
transferred, it may be advisable to sterilize the mouth of the vessel with an
electric incinerator (Fig. 2) instead of a flame.
4.5. Pre-culture treatments
Sometimes additional pre- or post-excision treatments are carried out before
transfer of the explant to the nutrient medium. In cultures of shoots excised
from balsam fir buds, higher survival rates and better morphogenesis were
obtained if the shoots were forced to bud break in an EDTA solution. Morphogene-
sis was also stimulated by a short soak of the explant in malonic acid, a res-
piratory inhibitor, before transfer to the nutrient (26). Surface sterilized
eucalyptus branch tips were treated for two hours in an ascorbic acid solution
and then stored in water before excision of the nodes (52). Embryogenesis in
coffee leaf explants was stimulated by keeping the explants in the dark on a
saline-sugar agar for 36 h before transfer to the nutrient (163).
4.6. Incubation environment
Many cultures grow well within a wide range of photoperiods and light inten-
sities. Illumination by fluorescent lights (Gro-Lux, or similar type) at about
1000 lux for 16 h per day appears to be satisfactory for most cultures (124).
Light quality is often important. In embryo derived callus cultures of Douglas
fir, red light stimulated adventitious bud formation (90). However, red light
stimulation of shoot induction is not universal; for example, in tobacco callus
cultures, blue is more effective than red in stimulating shoot production (158).
Green-yellow radiation repressed the growth of ginkgo pollen suspension cultures
(96), and spruce cultures grew better under Gro-Lux than under Warm-White fluo-
 25

rescent lights (129). Sometimes a dark pretreatment is beneficial, for example,

in embryo cultures of Pinus contorta where bud formation was stimulated by a
l2-day dark exposure of the cultures (196). In cell suspension cultures of jack
pine, free amino acid nitrogen decreased in cells exposed to darkness (57).
Fluorescent tubes emit a significant amount of near ultraviolet (300-400nm)
light (96, 192), ranging from 2.15% of total emission for daylight tubes to
1.44% for Gro-Lux tubes (8). Pyrex glass, which filters out all radiation below
300 nm (94), passes this near ultraviolet light freely, and therefore the medium
and tissues in glass culture vessels are exposed to this radiation. The near
ultraviolet has a pronounced effect on many biological systems (95), including
tree tissue cultures, where it inhibited the growth of cells derived from ginkgo
pollen (96). The near ultraviolet emitted from fluorescent tubes was found to
cause the formation of toxic levels of peroxides, and the destruction of ribo-
flavin, tryptophan, and tyrosine in the medium, and induced single-strand breaks
in the DNA of cultured cells (192, 193). Near ultraviolet is absorbed by flavo-
noids and quinones in the respiratory chain, and the energy is transferred to
DNA, thus enhancing mutagenic damage (195). Therefore, light quality should be
considered when choosing light sources for culture rooms, and intensity and
quality should be checked periodically because these change with time.
Most tissue cultures grow well within the temperature range of about 20-27C.
For optimal rooting, higher temperatures are sometimes required, an example
being cultures of grape vines which rooted best if exposed to constant 32C, or
to 39C for 2-4 days followed by exposure to 32C (62).
4.7. Transfer to soil
In many tissue culture cloning programs, one of the most difficult problems
is transfer of propagules from culture to soil. In most cases, the cultures
produce adventitious shoots which are excised and returned to a culture medium
for rooting, or are rooted in a soil rooting bed in a misting chamber. If
adventitious embryos are formed, they are kept on various culture media till
after they germinate and then are transferred to soil.
Some of the problems in the transfer of propagules to soil are: 1) The prop-
agules do not survive abrupt transfers. Growth regulators and organics should
gradually be withdrawn and light intensity gradually increased. 2) The prop-
agules desiccate after transfer. This can sometimes be prevented by using mist-
ing chambers or other means of maintaining high atmospheric humidity. 3) Damp-
ing off by fungi is common, especially in misting chambers, and special antifun-
gal treatments may be required. 4) By the time the propagules are large enough
26

for transfer, they may have become dormant and require a low temperature rest
period before transfer. For a more detailed discussion of these problems and
other technical aspects of transfer to soil the reader is referred to other pub
lications (114, 179).

5. CONCLUSION
Tissue culture of forest trees is rapidly entering a new era. From being
purely a research tool it is increasingly becoming an integral part of various
forest operations. Already it is applied in tree breeding, selection and propa'
gation programs, and the prospects are bright for its future use in tree diseas
control and the production of metabolites, drugs, and other valuable compounds.
For forestry application, it is essential to standardize and optimize the
tissue culture procedures as much as possible. Reliable methods that consis-
tently produce uniform results are needed because in large-scale commercial
applications even a temporary failure of the techniques is costly and therefore
not acceptable. As was outlined throughout this chapter, there are countless,
often unknown, factors that interfere with even the most standardized tissue
culture procedures. In the past, the main emphasis was on the establishment of
the major factors controlling growth in tissue culture. However, the point has
now been reached where the more minor controlling factors should also be deter-
mined and standardized to make large-scale research and commercial application
possible on a reliable basis.

6. ACKNOWLEDGEMENTS
I wish to thank Dr. W. K. Coleman, Canada Department of Agriculture,
Fredericton, N.B., Canada, for reviewing the manuscript.

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 33

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34

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 35

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36

3. CELL AND TISSUE CULTURE IN FOREST INDUSTRY

D.J. DURZAN

1. INTRODUCTION
The currently ava ilable body of knowledge of cell and
tissue culture of forest trees is mainly a product of
scientific curiosity rather than nec essi ty. Once the basic
techniques were established in the 1950's and applied to the
propagation of horti c ultural a nd agricultural plants,
managers enga g ed in tree improvement programs realized that
similar methods could even tuall y be u sed commercially in the
forest industry. However, although cell and tissue culture
was the strategic new technique needed to bring the study of
trees into the laboratory (10), tissues of many mature trees
r e main somewhat recalcitrant in response to most standard
practices . Furthermore, the technique coul d not progress
until:
A. Methods were availab le for the genetic selection
of trees, cells and tissues.
B. Synthetic plant growth regulators (e .g.,
a-naphthalene acetic acid, N6 -benzylaminopurine,
were ava il ab l e and proven effective in controlling
t he growth and development of a wide range of
spec ie s (Fig. 1).
C. Ce llular nutrition could be impr oved and control-
led through al l aspects of the propagation pro-
cess .
Shortages of food , energy, materials, shelter and
transportation have forced governments, private individuals
and commercial enterprises to intens ify the ir search for new
ways to sustain and improve the prof it ab ility of l oca l
 37

III
~
a:
w
D..
III
o
Z
BENZYLAMINOPURINE
~20
C)

~
III
iii
w
zw
C)
o
it 10
a:
o NAPHTHALENE ACETIC ACID
~
u..
o
~
a:
oD..
w
a: 10- 4 10-5 10-6 10-7 10-8 10-9
MOLES
FIGURE 1. The relationship between reports of successful
morphogenesis 6 reported in gymnosperms and the molar concen-
trations of N -benzylaminopurine, c(-naphthalene acid,
(3-indolebutyric acid (IBA) and f3-indole acetic acid (IAA)
added to nutrient media. Data are collected from the
literature to 1980 .

forests. Cell and tissue culture technology may improve

forest productivity by ensuring that:
A. Quality trees are selected and mass-produced from
our dwindling genetic reserves (41, 54, 58).
B. Risks associated with the collection and quality
of seeds for afforestation programs are reduced
(56)
C. The additional annual productivity per acre gained
through tissue culture can be multiplied by the
number of acres of forest land. For some agencies
this productivity becomes a very great multiplier
of profits (47).
D. New genetic variation can be introduced in clonal
materials, and the barriers to crossability among
species can be reduced using the powerful methods
of gene splicing developed in agriculture and
medicine (23, 42).
38

E. Disease-free clones can be produced to improve

plant vigor and productivity (34, 52, 86).
The forest resource is far from being fully domesti-
cated (58). The United States Department of Agriculture
(89) has estimated the increases in tree productivity that
can be obtained from tree genetics (Table 1). In vitro
systems increase our ability to select and test for trees
that will grow successfully and rapidly (52), are disease
resistant (15), and are responsive to common silviculture
practice without the need for nutritional amendments to soil
(8, 14).
Another aspect of tree-improvement programs is the need
to produce trees suitable for specific types of land-use
(80, 95), energy and material substitutions (4, 11, 12, 19,
59, 60), climates like arid and cold zones (5, 78), and
multiple uses (food, fiber, fuel, recreation and amenity
forestry) (91, 94). Cell and tissue culture technology can
also help the forester deal with the long-life cycle, size,
vulnerabili ty and complexity of trees. For example, costs
and risks in nursery practice are high because of the
seasonal nature of growth and development and the suscepti-
bility of germinating seeds to disease. Continuous propa-
gule production in vitro and subsequent field tests, associ-
ated with the certification of trueness to type and a
disease-free condition would help reduce these risks.
To achieve the above objectives requires innovation,
large-scale future scientific efforts, and a cost-effective
scale-up (20, 79). Unfortunately, the technology may suffer
from a "long lead time" and not be sui table where returns
are sought over the short-term (30, 66).
It is important to find the "best fit" for biological
technologies (64). Initially, multiple-use agri-forestry
systems (17, 90, 91) coupled with low technology, where man-
power (scientific and labor) is readily available, may offer
the best opportunity and short-term profitability. This may
be so in India, where the rural poor require fuel wood on
marginal lands, especially in arid zones (22, 37).
 39

Table 1. Anticipated genetic gain resulting from the first

generation of breeding and its effect when improved trees
are managed under intensive silvicultural systems (After
89) .

If
Character- genetic
istics gain Then the effect
Tree under is at may be an increase
species improvement least: in measure of:

Red pine Height growth 3% 300 million board

feet more per
rotation

White pine Resistance to 13% Double per-acre

blister-rust yields; 4 x acres
planted; losses to
rust in young
plantings greatly
decreased

Jack pine Form, height 11% Up to 70,000 cords

per year

Shortleaf Height 9% 1 million board

pine feet per year

White Volume 23% Nearly 2 million

spruce board feet more
per year harvested
and over 5,000
additional cords
per year

Black Volume 18% 90,000 cords per

spruce rotation

Yellow Apical 10% 84 million board

birch dominance feet more per
rotation

Black Diameter 10% Shorter rotations;

cherry growth 7,860 board feet
Apical 25% per acre more per
dominance rotation

For developed nations, where high technology may make

the difference in profits, industry can expect to benefit
from "in-house" skills, trade secrets, patents, and
40

international agreements, particularly with Japan where

large-scale plant cell cultures have been established with
tobacco (62). Here the key is to be first and stay first
through continuous high-quality research and development.

" 1-"
~e~
-"II
SEEDS Ij
~H

PROPAGANT:. ... ___ ~~

it
J'~t ~:~GRHv:~::~N>/~::.:e"~:~;;;G",,\
J. \ \
--

COMMERCIAL" } ~
PRIVATE \ BULK HEAT END
fEDERAL LAND , MATERIALS WASTES USE
l. ". ~
L---r-~ DOMESTICATION ,/' \ '
\ I ",' \
, /' DEFORMATION MANUFACTURING

' ..... , ,,""'"

- - " : _ _ _ ENVIRONMENTAL
'-~. /
& GENE QUALITY

PRODUCTION UTILIZATION

FIGURE 2. Renewable resources such as forest trees are

developed through the forest production cycle for bulk
materials that are used in manufacturing. In both cycles,
solar energy is needed.
2. PRODUCTION CYCLE
Figure 2 illustrates the basic components of production
cycles in an industrial forest. The genetic resources
contribute to the availability of bulk materials, which in
turn feed into manufacturing cycles. The nature of . the
genetic resources determines how and to what extent bulk
materials are used to meet client demands.
Production in future cycles may be established at
several levels. First, there is natural regeneration of
trees (54). Second, many domesticated trees may be produced
for stereotyped units of vegetation characteristic of
agri-forestry systems or variants (74) Here specific
genetic gains, such as rapid growth rate, disease resistance
and fiber quality are important. With proper breeding and
selection of trees, the crop rotation time can be reduced by
taking advantage of geography, sunshine and population
genetics, even on marginal soils, and risks can be low
(Table 2).
 41

Table 2. Strategies that lead to the maintenance or reduc-

tion of genetic variation of indigenous populations of
forest trees (modified from 56).

Low-risk strategies High-risk strategies

- limited plantations - extended plantations

- homogeneous environment for - heterogeneous environ-
production ments
- manipulate environmental - no possibility of ma-
factors nipulating environment
- use adapted populations - use of exotic species
- practice based on plant- - untested trees
ation and field trials
- short rotation (poplars, - long rotation time
eucalyptus) (hardwoods)
- apply experience with - ignorance
juvenile-mature tissues,
clinical variation - ecotypic specialization
- select for high heterozy- - less heterozygosity
gosity
- use production population - use breeding population

Third, the genetic resources have yet-undeveloped

potential based on the potential of cell cultures (34, 55,
84). Cell suspension cultures for conifers were established
in 1967-1968 on a scale of more than a liter by this author
(36) in the laboratory of F. C. Steward. Today, some
workers are contemplating the scale-up of cellular systems
of agricultural species using existing facilities originally
designed for industrial microbiology (23, 24, 62, 93).
Fourth, biomass production can be increased by trans-
formation of genes and their enzyme-forming systems at the
molecular level (51, 97). Cells continue to hold the secret
to the sequential production of nucleic acids (82), proteins
and cellulose. One fundamental unknown process is how cells
produce reactive chemical networks. This process has been
the subject of recent imaginative articles on the prospects
for future high-technology systems (11, 12, 21). If genetic
information become manipulated and exploited, then the slow
evolutionary processes in biological systems would be
accelerated significantly. We may be able even to recover a
few of the now-extinct tree species that in the distant past
42

gave us the oil, coal and other nonrenewable resources that

we are now so dependent upon.
Table 3. Constraints to the productivity of forest trees and nature of the research effort needed
to obtain a major effect (31).

Constraint Nature of Research Major Effects

1. Plant breeding and genet-
ic manipulation limited
by life cycle and complex
nature of trees.
Strengthen tools of genetic worldwide potential is immense.
manipulation: plant breed- Production increases from most
ing and "classical" genetics; lines of research expected in
cell biology; genetic stocks; 10 to 20 years.
vegetative propagation via
cell and tissue culture
methods; population genetics.

2. Biological nitrogen fix- Increase biological nitrogen Large potential worldwide.

ation to overcome limited
availability of nitrogen.
fixation associated with tree Results within 10-25 years.
crops; improve recognized
symbiotic association; attempt
!~i~~;ae~~~h t~;! i:~~g o~~:~ci-
nonlegumes; transfer fixation
capability from bacteria to
plants, trees, and microorgan-
isms.

3. Tree growth rate and form. Improve photosynthetic effici- Higher yields of trees, partic-
ency to depos i t more wood ularly in the marginal areas;
relative to the available N. substantial increases in poten-
Increase shade tolerance and tial yields after 15 years or
photosynthesis in major species. more.
Consider reducing photorespir-
ation. Select and develop
trees with greater assimilative
surfaces (roots, shoots) with
distribution of leaves to optim-
ize use of incident light, and
with superior cambial and wood
development.

4. Resistance to environment-
tal stresses so that trees
could survive and grow on
poorer soi Is and in severe
climates.
Improve resistance of tree crops Larger and more stable yields
to drought, temperature extremes, in 10 to 15 years. Trees can
deficiencies of acid soils, salt be grown in new locations.
tolerance. Develop rapid scre-
ening techniques, shorter rota-
tion, larger root systems, better
use of soil fungi. Introduce
tree-farming systems.

5. Pest management to over-
come losses due to in-
sects, diseases, and
fires.
Reduce preharvest losses due
to pests; integrate pest
management; utilize specific
contro l mechanisms, e.g.,
mixed species plantations.
Large and pervasive losses due
to pests can be minimized in
short run by adapting known
techno logy and in long run by
biological control techniques.

6. Weather and climate (es-
pecially where multiple
constraints on growth are
imposed by light, temper-
ature. and moisture).
Improve techniques for predic-
ing weather and climate and use
information to reduce weather
damage; develop baseline infor-
mation about inventories cor-
related with site and climate
through remote sensing.
Substantial risk reduction in
short run, especially for
nursery stock; site selection
can increase payoffs substan-
tially.

7. Management of forest soils
to maintain nutrient bal-
ance and minimize under-
story competition.
Improve management of soils to Annual production on some lands
increase productivity; soil can be increased by 150 to 200
classification; land clearing percent.
methods; correction of soil
deficiencies; maintenance of
desired soil characteristics;
sui table cropping systems and
technologies .

8. Irrigation and water man-
agement to minimize pro-
ductivity losses.
Improve management of water May markedly improve yields in
supplies; adjust tree-farming some areas and make capi tal in-
systems and irrigation for vestments more effective.
optimal supply to trees; adapt
operations to water availabil-
i ty; study transpiration re-
tardants and acid rain.

9. Fertilizer supplements and
mycorrhizal associations
to overcome nutrient im-
balances.
Improve cost/return ratios of New and more efficient fertil-
chemical ferti lizers: develop izers may have great effect on
new methods of producing slow- production on poor sites.
release nitrogen and phosphorus
fertilizer; develop new fertili-
zers tailored to forest condi-
tions; select species that util-
ize nutrients efficiently; study
nutrient cycling.
 43

10. Tree-production systems Improve integrated production Realize potential to 2X to 4X

to make best use of all
relevant factors .
systems, particularly for in- present production in tropics;
dustries in developing coun- more modest increases in mar-
tries; improve methodology for ginal areas.
identifying appropriate pro-
duction systems, including
multiple cropping; integrate
soil and water management:
optimize equipment-labor re-
lationships; develop baseline
data for forecasting and con-
trol as it relates to the manu-
facturing cycle and consumer
needs.

We must be able to recognize opportunities to develop

reliable and improved production systems for the long term
(Table 3). To do this, genetic maps should be prepared.
The effect of gene combinations on the control of growth,
differentiation and morphogenesis in artificial and natural
environments should be known. This information is essential
for deciding what options in population genetics and genetic
engineering are suitable for industrial forestry.
Currently, most research has focused on the large-scale
conversion of biomass for energy and as a chemical feedstock
using microorganisms and immobilized cells (24). In Canada,
with its vast natural forest resources, the industry is not
always geographically best located to exploit these resour-
ces and minimize transportation costs (61). To determine if
biotechnology could help in overcoming these problems in
agriculture and forestry, the Canadian Ministry of State
Science and Technology (65) has evaluated the importance of
cell and tissue culture methodology. Studies have already
started at the National Research Council on the genetic
engineering of yeast that could produce ethanol from the
spent sulfite liquors of pulp and paper mills.
In the USA, industrial forests make up 14% of the
commercial forest land, but have one-third in the
highest productivity class (83). Using current management
methods the average potential productivity of forest indus-
try lands is 88 cubic feet per acre per year, 12% higher
than the national average. Management decisions are made on
the basis of how they affect the total operation and not how
each particular unit is affected independently: production
and utilization cycles are only part of the consideration.
44

Spurr (83) points out that assuming reasonable govern-

mental policies towards the industry, economic consider-
ations will lead to more intensive forest management on
industrial lands over the next quarter century. This is
also supported by the figures presented by Hall for Inter-
national Paper (47).
However, in forestry, progress will be slow, largely
because of the complexity of trees. Progress will depend on
long-term commitments for uni versi ty and industry research
and long-term contracts. There seems to be a reasonable
role for a government-subsidized program of tax incentives
to encourage industry to undertake forestry res e arch to
improve forest production. The role of such research was
already appreciated by Bailey and Spoehr (10).
The upper limits of the production cycle has been
evaluated by Hall (47). He states that in a natural stand
of Southern pine on a site with an index of 75 to 80, we can
expect a yield of 30 to 40 cubic feet per acre per year.
With selective harvesting and stocking of the residual
stand, a yield of 40 to 60 cubic feet per acre per year can
be obtained.
Use of plantations with unimproved stock, as is
practiced with most temperate species, would yield for
southern pine volume values from 100 to 160. This level
represents what Hall regards as the minimum baseline against
which future developments must be measured. This is where
tree improvement enters. In managed plantations with
first-generation improved stock we can expect 120 to 190
cubic feet per acre per year. Productivity can be more than
doubled over five generations of tree improvement and if the
silviculturist can overcome the limits inherent in present
management techniques (Table 2).

3. GENETIC RESOURCES
According to Zobel (98), genetic improvement of forest
resources consists of:
A. Locating and using the correct species.
 45

B. Using the best geographic sources within the best

species.
C. Selecting and breeding the best individuals within
the best sources of the best species.
Cell and tissue culture contributes to the production of
genetically improved trees by in vitro mass propagation
methods and by the removal of barriers to crossability for
the creation of new hybrids (31, 44, 52).
3.1. Energy and fuel-wood species
The potential of biomass energy from forest residues in
the USA and abroad is impressive (1, 7, 68, 73). The global
demand for biomass relates to the needs of the more than
one-third of the world's population that depends on wood for
cooking and heating (5, 6, 22, 37). Eighty-six percent of
all the wood consumed annually in developing countries is
used for fuel; of this total, at least half is used for
cooking, and the supply is diminishing rapidly (37). As a
consequence, the Indian Department of Science and Technology
(New Delhi) is examining ways to plant woody species on
marginal lands for multiple use by the rural poor. Fortu-
nately, some species are suitable for vegetative propagation
and coppicing and thus can be rapidly mass produced (45, 46,
81)
For biomass production, the major objective is to
obtain the maximum growth of the most desired wood in the
shortest possible time at as Iowa cost as possible. Zobel
(98) estimates that on good sites, 60% of the genetic gains
captured by tree improvement will be in the form of volume
and 40% will be in improved quality characteristics such as
wood. He favors three strategies:
A. Increase the yield on already good sites.
B. Develop trees capable of growing on marginal or
nonproductive areas that currently will not
support an economical forest enterprise.
C. Develop trees with wood that has more Btus and
less moisture and ash or a higher chemical
content.
46

Of special importance is the work on biomass aimed at

obtaining valuable chemicals from wood and cellulose (29,
38, 70). Glucose derived from cellulose is a feedstock for
many fermentation processes besides yielding ethanol (24).
Lignin is another feedstock that has unexploi ted potential
(57) .
3.2. Multiple-use species
Some woody species provide fuel, food and quality
woods . Some adapt well to different sites and are easily
established. They require little care but should be protec-
ted from goats, cattle and wildlife. Furthermore, some
could assist in land reclamation, even when grown on steep
hillslopes, on low-nutrient or toxic soils, or in arid zones
and tropical highlands.
Special consideration should be given to such charac-
teristics as adaptability, nitrogen-fixing ability, rapid
growth (short rotation), ability to coppice, production of
wood of high calorific value that burns without sparks or
toxic smoke, and drought tolerance. Species falling in
these categori e s are Acacia, Gmelina, Eucalyptus, Alnus,
Albizia, Pinus, Calliandra and Prosopsis. One liability of
the more "weedy" species is their invasion of soils suitable
for agriculture.
3.3. Tropical legumes
The legume shows most promise for producing increased
supplies of vegetable protein that the world will need in
the near future (3, 7). Leguminous plants are found
throughout the world and the greatest variety grows in the
tropics and subtropics. A leguminous crop like Leucaena can
add up to 500 kg of nitrogen to the soil per ha per year
(3). Preliminary work with tissue cultures of Leucaena
seems to show promise for the vegetative propagation for
this genus (72).
Leguminous timbers (Baphia, Pterocarpus, Dalbergia)
have been in world markets for centuries and command high
prices in international trade (3). Most of these, however,
are slow-growing trees that have never been cultivated in
 47

plantations. Nevertheless, some of these, especially the

rosewoods, can be cloned by cell and tissue culture methods
(Mascarenas and Jagannathan, personal communication) and
thus may be used for large-scale planting.
As in agriculture, much of the anticipated gain in
forest productivity will depend on the availability of cheap
fertilizers, pesticides, and energy for irrigation and
mechanization. Energy is no longer cheap and neither are
nitrogenous fertilizers. Under the pressures of population
increases and energy and material substitutions, techniques
that can increase production without expending large
quantities of energy are assuming greater importance.
Interest in legumes and biological nitrogen is therefore
increasing.
3.4. Fiber and pulpwood species
Fiber and wood from rapidly growing plants (50) are the
raw materials upon which the paper industry is based. The
paper industry is one of the few energy-intensive industries
that has the opportunity to become self-sufficient in energy
and materials.
Wrist (96) has identified five technological develop-
ments that must b e taken into account when considering the
future supply of fiber:
A. Lumbe r and pulpwood inte gration of lumber and
pulpwood systems.
B. Increased use of wood as a fuel.
C. Growing differentiation between timber avail-
ability and economic accessibility.
D. Greater exploitation of hardwood species.
E. The emerging importance of plantation forests as
future sources of commercial lumber.
The bottom line in forest supply will be to increase the
economic incentives for plantation forests (forests that are
man-made) with carefully selected species and designed for
economic harvesting techniques.
In Japan, the following projects will be given high
priority (9):
48

A. Effective use of tropical hardwoods.

B. Breeding of suitable woods for pulping.
C. Comprehensive use of woods and non-wood plants
(e.g., kenaf, bamboo, agricultural residues).
Propagation methods for fiber and pulpwood species are
found throughout this volume.

Table 4. Comparison of properties of two breeding systems aimed at propagating trees. The
first system involves the need for a seed orchard and the other a viable cellu"'I"ar
cloning cycle. The latter assumes that one day trees may be regenerated from cells
Q3l.

Growth cycle Decades to centuries 25-26 hours

Size Up to 250 it high 50-100 micron diameter

10 acres of seed orchard 10 Ii ters of suspension

S~~~6 progeny per year) culture

Numbers to
1) det e ct mutation 10 ~ trees 10 7 cells
2) detect linke d traits 10 trees 10 3 cells

Time to produce seed or 8-13 years, minimum of 2-3 1-2 years estimated (10 6 /
soma tic embryo month) (somatic embryos)
~;~~~lO~r t~~~:S ?rnom se~~li~;:
chards over 16 years)

Seed pro duction predictabi Ii ty Var iable s e ed years Controlled production in vitro
on demand - --

Uniformi ty of genetics Variable, selection required, Homogeneous, low error fre-

c ontrolled pollination quency, easy rogueing, unex-
pected variability

Propagation Vegetative (cuttings), graft- Up to 500 copies by organo-

ing, rooting (sometimes genesis (tissue, organs),
difficult) millions by embryogenesis
(research needed to establish
this alternative)

Ploidy Haploids difficult to produce Haploids easily cultured but

more research needed

Flexibility in breeding system Barriers to crossability Apply methods of microbial and

molecular genetics; barriers
to crossability can be re-
moved; genetic engineering can
be attempted

Types of breeding system Selection most applicable All breeding systems can be
mutation Hybrids possible for certain exploited with cells and their
inbreeding species: other methods slow protoplasts; field testing
hybridization and require 8-10 year genera- r e quired for certification of
backcross tion times quality and "true to type"
selection

Cost to maintain system $1,500/year/hectare (1979) $1,500 per liter (est.)

Numbers required 570 x 10 6 / y9ar (Quebec) Not established

1o~ox 71~67~e~~e~~rl~~;~rio)
Columbia)

Requirements to produce 10 6 Viable seeds l3/cone Not applicable

trees Cones/ramet 125 Not applicable
Seedlings/rarnet 1,625 Not applicable
Plantable trees/ramet 541 Not appl~cable
Ramets 1,848
Ramets per acre 170 (l6 I x16 1 ) ~:ii:/i~ter 10 6
Acres 10-12 Laboratory space 2,000 ft 2

Goals Improved seeds in quantity Mass propagated elite hybrids

*cf. Proc. 17th Canadian Tree Improvement Association, Gander, Newfoundland, August 27-31, 1979.
 49

INOUCTK* AND '-':RIIII"VE UTAlilISHMNT AND

SlTU ... TIONS, _OUINTIAl
TESTING Of i>lIIOGENY

1{ }
STIIUCTURAl
MICROOAGANI" INTEGRATION IFEEDeACK AND OU ... LlTY
..... IC ... l DOMIN"'NCE , OISE .... SCREENING,
GIRDLING , WOUNOING , NUTRITIOfIIAl ,

""',"0::"0 ",.",...,.,
--"'--". =i:: ...
CLIENT
NEEOS

~ ~~~.::~: --~
HI(OETUIMINATlOfli

(MERISTU'MQOlfIEOI

GENETICINl'VT
'"OVEN , M... TUIIE SPECIMENS
P'ROTEIN5YNTHEII5,
'ACKAGING AND QUALITY

ICREENING OF
~Y

CALLUS~
INOUCTION

INI'UT (AUXINfCYTOKININI
CAUTIONS

_ JUVENILITY INTER'RETATION
,..
CHARCO ... L

AT GENETIC ('HASICI ANO

I'HYSIOLOGICAL LEVELS

_ EXTENT OF STAotfG CQFlAEL ... TIONS IN TAU

- I'HASEMAENNIAL STAGE l lONG LIFE CYCLE

GENETIC
ENGINEERING TliIAITI"'EIlAVE01

....-"5 ACHIEVEO'

FIGURE 3. Phases and alternatives in a vegetative propaga-

tion system for woody species. The various phases in the
sequence are identified at the top of the diagram from the
upper left to the right. The alternatives are a) micro-
propagation (partially organized systems) and b) the
production of plants from protoplasts and cells via callus
(unorganized cellular systems). PVP is polyvinylpyrroli-
done used to absorb phenols (After 31)

4. PROPAGATION SYSTEMS
In forestry, three levels of tree selection are in use
and controlled: the origin of wild seeds, seed production
areas, and seed orchards (58, 95) In seed orchards,
aggressive forest-tree breeding should be established to
produce new genotypes.
4.1. Seed orchards
Seed orchards will be a source of genetically improved
seed for the forest industry for years to come (41, 58, 88,
95, 98). Where trees of high value are scarce, vegetative
propagation can be used but the returns would have to
50

justify the cost. In the short run, returns may be greater

for woody landscape plants.
Ove~ the long run, cell and tissue culture methods have

greater potential for mass propagation than seed orchards

especially if the propagules can be developed in suspension
culture, where the doubling time of cells is a matter of one
to two days (Table 4). If morphogenesis were controlled so
as to recapitulate natural embryogenesis and capture genetic
gains, ready-made propagules may be available in quantity
all year around for container programs. Through tree
improvement and seed orchards, rotation time of production
forests may be shortened by 25 to 50% (28, 75, 76).
The field-test trial period for Douglas-fir clones is
approximately nine years (75). Replication of the same
genotype over several test environments lS useful for
estimating the total genetic variation and for evaluating
genotype-environment interactions.
4.2. In vitro vegetative propagation
Vegetative propagation is seen as a complement to
existing seed orchards. The propagation system must be
client-oriented, and the genetic gains must be identified
correctly. Figure 3 shows one system. The key steps are:
A. Determining of client needs and the genetic gains
to be propagated. This includes identifying
heritable traits that are essential or nones-
sential, balancing or counterbalancing, and
desirable or undesirable. The specific genetic
traits must be evaluated in light of their history
and value. Screening and testing methods for the
trait should be available or developed. The
economic gain and options in the systems should
also be known.
B. Introducing precultural factors unique to the
species to facilitate propagation. Barriers to
propagation must be identified, and steps should
be taken to overcome these. Steps can include
removal of apical dominance in shoots of mature
51
52

trees and pretreatments that convert mature

tissues to a more j.uvenile phase (77). A feasi-
bility study should reveal unexpected factors and
identify the conditions allowing morphogenesis.
C. Choice of cell and tissue culture or organ (meri-
stern) culture. The systems of choice will involve
partially organized explants or the unorganized
tissue route (Fig. 3). The choice will be deter-
mined by the number of required propagules,
success in generating callus, cell suspensions,
number of clones, and the trueness to type or
variety required in the product (53). The par-
tially organized route may be labor-intensive and
it requires a sequential induction of plant organs
(Figure 4). Nevertheless, Gupta et al. (45) have
estimated that more than 100,000 Eucalyptus plants
can be obtained by organogenesis from a single bud
within a one-year period. The genetic stability
of gymnosperm cells in vitro and under low levels
of growth regulators makes the unorganized route

FIGURE 4. Organogenesis in juvenile hypocotyl segments of

white spruce (Picea glauca) lacking an apical meristem
(After 25,26,27).

A. Germinating seed showing the seed coat (c) and

hypocotyl (h).
B. EScised hypocotyl expla12.-tss on nutrient agar with
N -benzylaminopurine (10 M).
C. After 50 days each hypocotyl segment produces
adventitious buds on the upper part of the
hypocotyl. If segments are upside-down, roots
will form at the top of the segment largely under
the influence of auxin (26).
D. Multiple elongated shoots from a single explant
that was divided after elongated needles and buds
had formed. Photo at 175 days (total) in culture.
E. Two shoots and 3 roots (arrows) have been induced
without further treatment.
F. Same as in 4E but 4 weeks later.
G&H. The 2 plants of 4E have been separated, and growth
in soil is shown after 76 days.
 53

attractive for mass propagation. However, the

control of morphogenesis (85, 92) in cell suspen-
sions of woody species is not yet complete (31)
(Fig. 5).
D. Quality control and packaging of the product once
plant lets can be regenerated. Embryos may be
pelleted by several coatings to form either
artificial seeds that can be easily handled for
mass planting (e.g., 67) or embryos surrounded
with diploid nutritive tissue (Fig. 6; 87).
Alternatively, plants can be distributed in
containers for field planting, for evaluation of
trueness to type, and for total genetic variation
and genotype-environment interactions.
E. Physiological preconditioning of the field for
woody species. Control over nursery disease is
also important. This may be achieved by placing
fungicides in the pellet around the somatic embryo
or propagules. Disease-free certification by a
pathologist may be required (86).
F. Field testing and establishment of the propagules,
which require selecting the ideal genetic blends
for specific sites and end uses (75). We must
understand the relative importance of genetic and
environmental variation wi thin trees, betwecen
trees wi thin stands, between stands and between
geographic regions (58).

5. CELLS FOR CO~mERCIAL PURPOSES

In basic forest science, the application of new
technology depends on its fit within time and money prior-
ities: profit is the common ultimate yardstick. Unfortun-
ately, there is a difference between test-tubes and fermen-
tation vessels, pouring things and pumping them, and between
surface/volume ratio effects and mixing effects. Ignorance
of these still costs industry much in hard cash. Further-
more, although science is international and public, technol-
54
 55

FIGURE 5. Control of morphogenesis in cell suspension

cultures of Douglas-fir (After 31).
A. Mature Douglas-fir at The Institute of Paper
Chemistry served as a donor of bud tissue. Buds
(arrow) from lateral shoots from the upper crown
were placed on agar plates containing a MS
(Murashige-Skoog 1962) medium with 5.0 ppm NOAA
and 0.1 BAP under aseptic conditions to produce a
proliferating callus. After 3 months the callus
was placed in rotating liquid cultures in low
diffuse light at 20C to produce a fine suspension
of fir cells.
B. When cell suspensions were screened to collect
cells between 60 and 130 M in diameter and
appropriate stimuli were given, cellular growth
was trans .f ormed into new patterns resembling
somatic cell embryogenesis. The start of this
type of development is indicated by the
enlargement and growth of proembryo-like clumps in
the suspension (arrow) . Magnification 250X.
Figures 'B' to 'J' represent somatic embryos from
3-year-old donor trees.
C. Pro-embryonic clusters of Douglas-fir cell after 3
to 4 weeks of growth. Magnification 275X.
D. Each compact cluster continues to grow to produce
globular embryos with coherent cellular patterns
and tightly connecting daughter cells. At this
stage the 5-to-6-week-old embryos readily produce
chlorophyll. Magnification 2BOX.
E. Individual globular embryos become polarized in
their growth to form a shoot-root axis and show
signs of internal cellular differentiation. The
dark central core of cells is destined to produce
a vascular cambium (v). Magnification 290X.
F. Well-developed 3-month-old embryo from cell
suspension cultures illustrating the shoot-root
axis, emerging cotyledons (c), internal vascular
area (v) and well-defined linear arrays of
epidermal cells on the surface (e). Magnification
200X.
G. Longitudinal section of tissue from an emerging
shoot induced in response to a specific auxin/cy-
tokinin ratio. Shoot apex (sa); cotyledons (c).
Magnification IBOX.
H. The structure in 'G' before longitudinal section-
ing illustrates the surface view of cotyledons or
leaf-like structures (c) and shoot apex (sa).
Magnification IBOX.
I. Development of cotyledon-like structures (c) on
the surface of 6-month-old embryos while growth
continues. Magnification 150X.
J. Scanning electron micrograph of organized growth
patterns in a cross-section of a putative somatic
embryo. The growth, much like that of a
56

sphaeroblast, occurred in a highly organized

pattern to produce a pith (p), vascular cambium
(ca), parenchyma (pa) and an epidermal layer (e).
Magnification 300X.

ogy is seldom either. In industry, the aim is to get results

to the interested user quickly and to capture the market.
The better the scientific work is, the less likely it is to
be published. For this reason, industry should employ
quality scientific personnel to recognize which new scienti-
fic developments are suitable for commercial exploitation.
5.1. Creation of new hybrids
A wide range of promising traits cannot be introduced by
sexual crossing because of natural crossability barriers.
Evidence now suggests that some barriers may be overcome by
genetic engineering with protoplasts (Fig. 7; 40, 44). For
our purposes, genetic engineering can be defined as any
nonconventional method of genetic manipulation dealing with
transfer of genes between trees and from other organisms to
trees. The modification occurs at the molecular and cellular
levels and even can be done with somatic haploid, diploid, or
polyploid cells, i.e., non-sexual cells.
Present methods permit new genetic information to be
inserted directly into the protoplast (the egg itself is a
protoplast, which after fertilization becomes multinuclear).
It is even conceivable that individual protoplasts can be
fused with haploid nuclei so as to establish a spectrum of
heterozygosities for a range of enzymatic loci.
Genetic engineering with plant cells is dependent on the
phenomenon of totipotency (85). In totipotency, live cells
are able to express all genetic information needed to recon-
struct the parent plant. The stimuli to reconstruct plants
from cells and tissues are known for 200 plant species (67).
So far several woody species have been grown from cells
(e.g., 31, 35, 52). The time is ripe for exploitation of
genetic engineering with forest trees (31, 34).
 57

FIGURE 6. The development of suspended cells into clumps

wi th internal meristemoids. Meristemoids eventually grow
through the initial structure much like a germinating spore
in a quarter strength MS medium low in calcium and supple-
mented with 0.1 ppm NAA and 0.01 ppm BAP (Hwo and Durzan,
unpublished data).
A. Cross-section of a clump (ca. 0.6 rom long) 2
months after culture showingthe meristemoid (m)
with cytoplasmically dense cells having large
nuclei. Cells around the meristemoid appear
digested (n).
58

B. For comparison, a cross-section of a developing

seed showing the young embryo (e) embedded in the
nutritive haploid female gametophyte (g) (courtesy
H. Kaustinen). Magnification X 350.
C. At 6 weeks, a light microscopic view shows that
the spherical clumps (ca. 0.2 mm in dia.)
contained dark green centers, which represent
internal meristemoids. The number of meristemoids
per clump is usually one but this can vary,
depending on auxin and cytokinin levels.
D. After 3 months, the internal meristemoid grows and
develops at the expense of surrounding tissues and
eventually ruptures the surface cells to enterge as
a white compact mass of cells (ca. 1 mm long).
These cells are derived from cotyledon callus of
Douglas-fir.

FIGURE 7. Loblolly pine protoplasts with single and multiple

nuclei grown on a modified MS medium under diffuse light at
20C (courtesy J.Litvay and D. Verma). c, cytoplasm; n,
nucleus. X 470.

A. Cross section of a protoplast with a single

nucleus.
B. Protoplast-fusion product with 2 nuclei from the
same line.
C. Protoplast-fusion product containing 3 nuclei.
 59

5.2. Biochemical transformations with cells and enzymes

Biochemistry, as developed from the time of Leibig and
Pasteur, is rooted in industry and emerged through fermen-
tation and the production of pharmaceuticals and petrochem-
icals (49). Cells are used as catalysts to convert raw
materials and waste materials to a more sui table form for
energy (biogas, alcohol), food, and animal feed (single cell
protein, etc.) (2). Cells can be used to study mineral and
chemical cycling (8), mycorrhizal interactions, the control
of soil-borne pathogens and symbiotic and asymbiotic
nitrogen fixation (23).
As of now, the use of tree cells to produce antibio-
tics, vaccines, and secondary metabolites is limited.
Experiences with cells from other plants suggests that some
organization and morphogenesis may be needed to produce the
products of value (84). Results of some studies suggest
that plant cells in the stationary phase behave much like
cells in leaves (Gnanam, University, personal communi-
cation). Conifer cells grown in vitro may have less than
the haploid number of chromosomes (71). The use of
hypohaploids with a fraction of the genome for the
production of secondary products has not been explored.
"The integrated use of biochemistry, microbiology, and
chemical engineering, to achieve technological (industrial)
application of the capacities of microbes and cultured
tissue cells" (69) is emerging. Cells have been immobilized
in columns or grown in fermentors for the production and
conversion of carbohydrates, lignin and lipids (18, 29, 57).
Radiation-induced immobilization of chloroplasts prolonged
oxygen evolution and improved the thermostabili ty of the
preparations (43). Enzymes have been retrieved from
organisms such as bacteria growing in hot springs (80C).
This has improved the stability in immobilized enzyme
processes (62). It is here that Japan and the USA (48, 51)
are collaborating to rapidly develop industrial
applications.
60

Through gene-splicing, cells may be altered to produce

in quality specific products in fermentors, such as lectins,
specific hormones, and drugs. Stanford University, where
the arcane new science of gene-splicing was developed, has
begun to license the use of its patented technique (US-4,
273, 224). Charges include a sign-up fee of $10,000 (US
1981) and $10,000 for each successive year. The royalties
range from 0.5% to 1 % of product sales. According to the
Wall Street Journal (August 4, 1981), some 200 US companies
are believed to be using the patented technique (US-4, 237,
224). We have yet to see the first commercial product of
this technique.
Biochemical engineering with cells (24) offers advan-
tages over traditional chemical engineering in that lower
temperatures and pressures can be used. Some disadvantages
are:
A. Genetic instabilities, especially of microbial
cells.
B. The lack of methods to immobilize cells for
continuous productions.
C. Water is required in large quantities. Cells may
not tolerate toxic organic by-products that build
up with time. Hence, purging systems are needed
to remove the build-up of metabolic products.
D. Problems in the scale-up of engineered organisms.
Tree cells do not yet have a role in industrial
processes, because only small quantities of the products are
produced internally. The productive periods are difficult
to maintain longer than one or two weeks. In some instan-
ces, cell suspensions of conifers, such as white spruce and
jack pine, have been maintained for over a year through
biweekly or monthly subculture (Durzan unpublished). Other
studies of plant cells in suspension have explored leaky
mutants, the control of cell division, morphogenesis and
genetic modification (42, 55).
One interesting application of a product produced by
cell suspensions would be the use of lectins to attach
 61

FIGURE 8. Under stressful conditions and in germ-free or

contaminated cultures, conifer cells release lectins to
produce a cloudy medium.
A. Aseptic culture of Loblolly pine produces in-
creased medium clouding in response to increased
sucrose (left to right 0, 0.03, 0.1, 0.3 and 1%
sucrose).
B. Scanning electron micrograph of a clump of
suspended conifer cells showing bacteria (arrows)
entangled in cell wall material, which is
believed to contain lectin (3300X). The binding
of microbial cells to plant cells and organs
offers a new system in agricultural microbiology
(13, 23).

N-fixing microbes to leaves or roots of trees (Figure 8).

These microbes would slowly release nitrogen for sustained
62

growth of trees. The lectin required would be obtained from

cell suspension cultures that have responsed to the
microorganism. The study of infection of legumes by
Rhizobia should shed light on how to employ this concept
(13, 23).

6. CONSTRAINTS
There are still many yet unproven culture systems and
recalci trant species, especially for more valuable forest
trees (50). We must ask why the current technology does not
apply and how to foster greater success. This concern is
important because plant cells are far more complex than
microbial cells. We still have a long way to go in
controlling the reactions in our culture systems (42).
We cannot yet prescribe the rules that limit the
capacity of plants to fabricate biomass. Even when there is
no organ ized shoot tip or meristem or no newly generated
engineered genetic system, the tissue culturist knows that
cells may still respond metabolically to the external
variables of light, temperature and nutrition. Better
control over organized growth and morphogenesis is of high
priority and a prerequisite for the application of genetic
engineering technologies can be applied to cells of woody
species.
Difficult problems ln cell and tissue culture and their
associated technologies still limit the application of these
methods on a large scale. For example, in the genetic
e ngine er ing of plant cells, the efficiency of gene transfer
and express ion is not yet very high and is restricted to a
few host systems (42) . For production purposes the genetic
stability of the cloning vehicles themselves is unknown.
Whil e landmarks in engineering have been achieved with
bacterial systems, the transfer of a set of 17 nitro-
gen-fixing genes to a more complex cell such as yeast did
not lead to nitrogen fixation (23). Gene transfer is
clearly not enough. Furthermore, plant storage protein~ are
usually a collection of subunits, each coded by genes that
 63

may be highly separated from one another and that have

multiple expressions. Clearly, the system may be more
complicated than first suspected. In addition, plant cells
tend to accumulate metabolic products. Hence, "leaky
mutants" or "release factors" will have to be sought if
continuous production processes are required.
Where increased productivity is postulated on the basis
of new cellular associations, the host cell may have to be
modified substantially before productivity can be realized.
For example, when leaky mutants of nitrogen-fixing bacteria
are fixed to the roots of a species such as corn, the plant
cannot support this type of association. The claim is that
corn should be bred to produce sufficient photosynthate to
meet the excess demands of arising from the nitrogen
released by the bacteria (23). However, if the current
productivity is exceeded, new threshholds may introduce
other factors for which we may have insufficient knowledge
and limited control.
At the genetic level, when protoplasts from two
different plants are fused and where a hybrid plant can be
regenerated, the hybrid such as that of a potato and tomato
bears fruit that are a blend of characteristics rather than
separate entities, such as potatoes in the ground and
tomatoes hanging on stems (63). More product specificity is
needed if such technology is to be applied to biomass
species.
We cannot yet guarantee that trees propagated through
cell and tissue culture will always be uniform or responsive
to past methods of cultivation (53). For the more
commercially important woody species, the excised tissues
tend to remember their original position and age in the tree
and continue to express this potential even when separated
from the donor (33). For reasons unknown, genetically
identical propagules may develop differently either
physiologically or morphologically and this may add to the
overall variability. Until cell and tissue culture methods
64

can be applied to older and genetically proven specimens,

the utility of this method will remain limited (77).
Once woody species can be mass-produced, we cannot be
sure how well the plantlets will be transferred from the
tissue culture chamber to the field. Where physiological
and nutritional preconditioning is required, the transfers
to the field may be difficult on a large scale especially
where the propagules may be thrown from the soil by frost
heaves or erosion. Nevertheless, facilities that can deal
with millions of plants at one time are already available
especially for coniferous species and some horticultural
varieties.
Also at issue is whether tissue culture can produce
biomass at a reasonable price. For the boreal forest the
aim is to produce a propagule at a cost of one or two cents.
Ornamental and fruit trees can absorb much higher costs.
For easy-to-root hardwood species, such as red maple or
locust, the production cost per 1,000 containerized and
improved seedlings would be approximately $125 (cf. Brown
and Sommer, this volume). Even if production costs seem to
be within reason, costs must be determined over the entire
cycle of cultivation and harvesting. New water conservation
and harvesting techniques are needed to get the maximum
fiber and fuel from biomass plantations (5, 96).
We must determine whether agri-forestry systems can be
established through tissue culture to me e t the challenging
needs of man. In third-world countries, multiple-use
agricul tural and forestry systems with short rotations are
being sought . The hope is that use of tissue culture
methods in these systems will help compensate for the needed
controls over environmental factors, insects, disease,
nutri tion, and man's intervention to produce biomass for
unproductive lands (6, 39). Because there are currently no
sound, well-established scientific generalizations for
determining in advance the best cultural treatment for a new
unit or mix of vegetation, we must look to countries with
 65

natural units of forest vegetation to help us avoid mistakes

in the future.
7. OUTLOOK
Confidence in cell and tissue culture technology has
been slow in developing. In some cases confidence may be
lost because of unrealistic promises and unfulfilled ex pect-
ations. Nevertheless, progress has been encouraging, and
ventures large and small are being established. A new
International Bio-energy Directory (16) lists activities in
60 countries including 1,850 different projects. Some of
the projects are already employing tissue culture, including
those of a few tree species.
In France, the Association For~t Cellulose (AFOCEL) has
been engaged in the in vitro propagation of Douglas- fir,
Pinus and Eucalyptus sp. In the next few years field trials
of propagules will establish the value of the technique. In
older specimens, the problem of maturity may be overcome by
successive graftings of the older material onto young
rootstock before in vitro propagation is possible (33).
Seed companies are starting to study the commercial
mass production of trees, and oil companies have diverted
profits into bold ventures involving the genetic engineering
of plant cells. Cetus, Inc., at Madison, Wisconsin, with
the support of funds from the pulp and paper industry, will
establish in 1981 the first genetic engineering venture
aimed at agriculture and forestry to the year 2000.
The great problem remaining with cells is how
engineered DNA and the potential of plant cells are
expressed through the cell cycle, episodic growth of the
tree, stand rotation, and the long life cycle the species.
We are only just starting to explore the role of the
organization of cytoplasm and of cells in tissue
hierarchies.
In the short-term, the next 10 years, the only major
application may well be the clonal propagation of trees
largely for agri-forestry and multiple-use systems and
primarily in underdeveloped countries. In the United States
66

and Canada, the process may take longer. We will have to

consider species that offer specific alternatives in
material or energy substitutions and the development of
rapid screening methods for quality and disease-free
certification of our forest trees.

8. ACKNm.oJLEDGMENTS
The author thanks the Institute of Paper Chemistry for
the use of Figures 2, 3, 5, 6, 7 and 8.

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72

4. IN VITRO PROPAGATION OF GYMNOSPERMS

A. DAVID

1. INTRODUCTION
Most gymnosperms are propagated sexually through seeds. With regard to
genetic improvement of a species, sexual reproduction will preserve some of the
mean traits of selected families. Vegetative propagation, on the other hand,
is a procedure with the potential to maintain the best characteristics of se-
lected individuals, and therefore often is the preferred method of propaga-
tion.
Vegetative propagation generally involves the formation of new meristems
from differentiated tissues. If cuttings are kept under favorable conditions,
they sometimes can be transformed into a new organism, possessing the charac-
teristics of the parent individual.
In spite of extensive experimentation during the last several years, many
aspects of vegetative propagation remain poorly understood. The age of the
tree from which the cuttings are taken, their position on the tree, the time of
their removal, and the seasonal variations in rooting capacity are all factors
in vegetative propagation that require further study. Vegetative propagation
of many species, especially those of the gymnosperms, is still a major problem
to be solved.
The establishment of continuous cambial cultures as reviewed by Gautheret
(38) gave a new stimulus to research.
Research with several herbacious plants has shown that whenever it is pos-
sible to maintain cell multiplication of a callus, one can, under certain nu-
tritional conditions, initiate the formation of organs. For some species,
plants can be regenerated from single, isolated cells in culture (51), or hap-
loid plants can be obtained from anthers in culture (55). Sometimes "vegeta-
tive embryos" are produced with various types of cultivars (56).
Although the first research in tree tissue culture was carried out a long
time ago, it did not incite much enthusiasm initially . It was not till 1950,
that work with gymnosperms started seriously, probably because of encouraging
results obtained by Ball with Sequoia sempervirens (4). Since then, tissues of
 73

many gymnosperms have been cultured, resulting, in some cases, in the isolation
of continuous cultures, and occasionally in organogenesis. In 1975, Brown and
Sommer abstracted all the work carried out with in vitro cultures of gymno-
sperms (14).
This chapter is based largely on a review published by David and Thomas
(33), updated with more recent information. The first part will deal primarily
with organogenesis in callus cultures. Direct morphogenesis in organs or organ
sections will be detailed in the second part. The establishment of plants from
vegetative material obtained from mature specimens will be discussed. The
various publications dealing with each of these will be discussed in chrono-
logical order (Bibliography ending March 1981) and summarized. Finally, a few
new working methods, arising from this survey, will be presented.

2. ORGANOGENESIS IN CALLUS AND SUSPENSION CULTURES OF GYMNOSPERMS

Evidence for morphogenetic activity in callus of Sequoia sempervirens, car-
ried through several subcultures, was presented by Ball in 1950 (4). Following
this event, Jaquiot (43), working with cambial explants of Abies nordmanniana,
established the shoot forming effect of adenine-like substances, in contrast to
the root forming effect of auxins.
Norstog and Rhamstine (58) cultured proembryos of Zamia integrifolia, and
female gametophytes of Cycas circinalis, and obtained callus in the presence of
2,4-dichlorophenoxyacetic acid (2,4-D); transfer of the new tissues to a nutri-
ent low in hormones allowed the formation of adventitious embryos.
Callus originating from hypocotyls of Picea abies provided cell suspensions
when kept in a liquid medium. Chalupa and Durzan (20) noticed the formation of
proembryo-like structures in these cell suspensions.
Konar (47), working with Pinus gerardiana observed the formation of buds and
roots in cell colonies derived from hypocotyl cultures.
Shoots were induced by Cheng (23) in callus cultures derived from cotyledon
tissues of Pseudotsuga menziesii. The cotyledons were cultured in a medium
containing 5 ~mol abscisic acid (ABA), indolebutyric acid (IBA), benzylamino-
purine (BAP) , and 2-isopentenyl purine (2-iP). The initiation of shoot meri-
stems was subsequently obtained by adding to the surface of the cultures a so-
lution of highly concentrated BAP (0.5-1.0 mmol). The morphogentic activity of
the cultures was maintained by repeating the BAP treatment after three or four
subcultures carried out at three- or four-week intervals.
74

Embryoid formation was obtained by Ramawat and Arya (59) in callus of

Ephedra gerardiana on a medium supplemented with BAP and kinetin, with or
without auxin. Leafy shoots developed if the nutrient contained only kinetin;
auxin, on the other hand, favored root formation.
Reilly and Brown (62) cultured leaf sections from juvenile seedlings, or
embryos removed from unripe seeds of Pinus radiata, in nutrients with auxin and
cytokinin and obtained a compact callus. After transfer of this callus to a
medium free of growth regulators, shoots were initiated after 12 days.
Chalupa (19) cultured embryos of Picea abies on a mineral medium with IAA
(25 mg 1-1) and kinetin (0.5 mg 1-1), and embryos of Pseudotsuga menziessii
with naphthaleneacetic acid (NAA) (1-2 mg 1-1) and BAP (1 mg 1-1), and ob-
tained unorganized chlorophyl-rich outgrowths. After 12 weeks several buds
were formed on the callus.
Winton and Verhagen (77) obtained callus from cotyledons of Pseudotsuga
menziesii in the presence of BAP and naphthoxyacetic acid. Shoot formation
occurred in 31% of the cultures.
Root and shoot formation occurred in cultures of Biota orientalis (Fig. 1)
in the presence of NAA, and zeatin or kinetin (68). This capacity for organo-
genesis was maintained after subculture.

Figure 1. A callus culture derived

from excised hypocotyls of Biota
orientalis. Note the development
of buds (~) and organs resembling
roots (4-). These cell colonies
have been subcultured three times,
at monthly intervals (Thomas, Per-
sonal Communication).

Webb and Street (76) found that cultures of embryos removed from unripe
seeds of Pinus contorta and Picea sitchensis' developed callus with a shoot
forming capacity on a medium with a mixture of auxins and cytokinins (IAA, IBA,
2-iP, BAP). This capacity for shoot formation was maintained after transfer to
fresh medium.
 75

Reilly and Washer (63) found that cotyledons of Pinus radiata, as long as
they were in contact with a shoot inducing medium, produced a callus with
meristematic cells that formed buds. The calli remained mitotic for several
months while retaining their shoot forming potential.
Groups of cells resembling the first stages of embryos were observed in cell
suspension cultures of calli developing from needles of young seedlings of
Pseudotsuga menziesii and Pinus taeda (78). Precursors of indoleacetic acid
oxidase favored embryoid formation (45).
Kadkade and Jopson (46) investigated the effect of illumination on shoot
formation in callus from embryos of Pseudotsuga menziesii. Callus formation
was stimulated by wavelengths of 550 and 660 nm, and five times more buds were
formed in cultures exposed to 660 nm light (0.42 m W/cm2) than in those kept
in the dark. The authors are of the opinion that light stimulates shoot initi-
ation, but not elongation.
Durzan (34) reported globular embryos in cell suspension cultures of
Pseudotsuga menziesii shoot tips from 3-4 year old saplings from the green-
house. These structures became polarized along a root-shoot axis, and eventu-
ally developed cotyledons (Fig. 2).
Embryos of Pinus wallichiana cultured on a modified Murashige Skoog medium
containing NAA (0.1 ppm) gave rise to calli that were regularly subcultured.
Transfer of these calli to a medium containing BAP (1 ppm) resulted in bud
initiation. After replacing the cytokinin by coconut milk these buds elongated
into shoots (49).
Following the establishment of gymnosperm tissue cultures, the first mani-
festations of organogenesis, as described by Ball and later by Norstog and
Rhamstine, did not arouse the interest that these observations deserved. Many
years passed before further studies in this area were initiated and encouraging
results were obtained.
A survey of the literature indicates two categories of development in callus
and suspension cultures. In the case of callus, the exact origin of the new
shoots is not precisely known especially because histological studies were
generally not carried out. The original explant may have had an effect on the
formation of buds in the unorganized, newly formed callus.
Several authors have noted the retention of organogenetic potential after
subculture. Such investigations should form the basis for further studies to
determine if this morphogenetic potential can maintain itself indefinitely, or
whether it diminishes progressively in successive subcultures (most experiments
76

have not been carried out beyond a few subcultures). Whatever the outcome, the
induction of organogenesis in unorganized callus still appears to be only par-
tially achieved. Minocha (54) has attempted to solve this problem by exposing
callus, obtained from embryos of Pinus strobus, to more than 60 combinations of
auxin and cytokinin. However, this did not result in the formation of buds or
roots.
Therefore, unlike many herbacious angiosperm callus cultures, those of
gymnosperms have so far generally not expressed the organogenetic potential of
the cells. However, the fact remains that if we could obtain organogenesis,
either in unorganized callus associated with the original explant, or in cell
colonies that have gone through several subcultures, the door would be opened
to a new way of producing unlimited numbers of propagules (Figs. 1 and 2).

c
Figure 2. Formation of structures that possibly could develop into embryos in
suspension cultures of Pseudotsuga menziesii (Durzan).
A. Globular stage; B. Polarized development; C. Development of cotyle
dons (v = vascular region; c = cotyledons; e = epidermal layer).

However, genetic stability will have to be assured during the various phases of
regeneration of plants from unorganized cell masses. Meanwhile, one should not
reject multiplication directly from the original explant, because at present it
is still the most effective method of propagation of existing genetic traits.
 77

3. MORPHOGENESIS IN CULTURES OF ORGANS AND ORGAN SECTIONS

In spite of the potential benefits of organogenesis in cultures of unorga-
nized cells, most researchers have focussed their attention on the regeneration
of plants via a process which, though not always strictly ensuring genetic
uniformity, at least avoids the prolonged phase of genetic instability which is
encountered when the route or regeneration from unorganized cells in culture is
followed.
Therefore, in this section, we will discuss studies that deal with
axillary and adventive bud formation, embryogenesis, shoot formation, and
rooting.

3.1. Axillary bud formation

Islands of meristematic cells, or in some cases vegetative primordia, may
persist in the axils of leaves during the ontogeny of shoots. In the follow-
ing, studies will be discussed that describe the in vitro development of buds
capable of forming shoots from such persistent meristematic areas in the
plant.
Boulay and Franclet (9, 12) cultured dormant buds of Pseudotsuga menziesii
in the presence of 2,4-D(10- 6 mol), and obtained callus as well as axillary
buds.
Buds appeared in the axils of cotyledons and juvenile leaves of explants of
Pinus pinaster (Figs. 3 and 4) cultured by David and David (31) in a medium
containing BAP (10- 5 mol). By culturing brachyblasts with one pair of
needles these authors obtained bud development from the apical dome located
between the needles (Figs. 5 and 6).
Bonga (6) used shoots of Abies balsamea excised in the winter from buds of
15- to 20-year-old trees. In some cases the explants were soaked for various
lengths of time in water with N-dimethylaminosuccinamic acid, or 1-phenyl-3-
methyl-5-pyrazolone. The elongation of the shoot and the development of
axillary buds at the shoot apex or base occurring when the explant was soaked,
suggests that inhibitors were removed in the soaking process.
David et al. (32) and Isemukali (41) induced buds in explants of Pinus
pinaster hypocotyl sections with an apical meristem and cotyledons, collected
from 20 day old seedlings. The cuI ture medium contained, BAP (10- 5 mol)
and NAA (2.5 x 10-8 mol). After 15 days exposure to the hormones, well
organized axillary buds were formed (Figs. 3 and 4). In comparing the effect
of various mineral solutions, the authors noted a stimulatory effect by 15
78

mEq 1-1 of potassium in the medium. The initiation of the buds by exogenous
cytokinin was stimulated by the presence of cotyledons.
Rancillac (60, 61) using similar explants of Pinus pinaster, showed that
addition of lAA, IBA, or NAA did not stimulate the growth of cytokinin induced
axillary buds. On the contrary, the auxins stimulated callus formation. Gib-
berellic acid also had a negative effect. On the other hand, a medium rich in
minerals (like in a Murashige Skoog medium) stimulated organogenesis in the
explants.

Figure 3. Bud development (~) in the axil of cotyledons and juvenile leaves
of Pinus pinaster (David).
Figure 4. The same explant (Fig. 3) after a few weeks in culture ; note the
development of several shoots from the axillary buds .
Figure 5. A brachyblast of Pinus pinaster with one pair of needles shortly
after transfer to the nutrient and before transformation of the vegetative apex
to a bud (David) .
Figure 6 . A bud (~) formed at the apex of a brachyblast under the influence of
a cytokinin-auxin combination .
 79

Faye and David (unpublished) observed that a concentration of BAP as high as

2.5 x 10- 5mol, and a duration of exposure to the hormone as long as 45 days
was in some cases required for morphogenesis, depending on the degree of
reactivity of each explant. A large variability in axillary bud forming
capacity was observed between explants, with the number of buds being induced
by one cytokinin treatment varying from 4 to 37 per explant, the most common
number being from 10 to 20 buds.
Boulay (10) studied axillary bud formation in stem sections of Sequoia
sempervirens and found that the origin of the material had a considerable
effect. If the explants were taken from branches of a tree more than 20 years
old, no buds were formed. On the other hand, explants taken from stump sprouts
were very active. The author also noted a difference between explants taken
from current sprouts and those taken from the current growth on older sprouts,
wi th explants from the former showing a very high rate of bud formation.
Therefore, these studies demonstrate the relationship between organogenetic
capacity of the explants and the physiological age of the explant source.
Axillary shoots were induced by Cornu (Personal Communication) in current
shoots of Larix x eurolepis excised shortly after budbreak in the spring, and
grown on medium containing Murashige and Skoogs' minerals, and BAP, and IAA at
1 mg 1- 1

Thus, several authors working with various species have induced the
formation of axillary buds in vitro. The reacting cells are resting, primary
meristematic cells located at the axils of the cotyledons and juvenile leaves,
or at the apex of brachyblasts (Figs. 3, 4, 5, and 6).
Because systematic studies of mineral nutritional requirements are lacking,
it is impossible to recommend specific nutrient formulas. However, it appears
that high potassium promotes morphogenesis. Amongst the cytokinins that are
generally used (kinetin, zeatin, and BAP), BAP is the most active one if used
at concentrations of about 10-5 or 5 x 10-5 mol. An auxin (NAA) is often
used at the same time at about 10-8 mol. The hormonal combination is applied
over a period, which, depending on the concentrations used, varies from a few
days to a few weeks.
The intensity of the response depends on the physiological state and the age
of the parent at the time of explant excision. Presumbly all explants have the
same morphogenetic potential, regardless of the age of the material, but in
explants from mature trees this potential remains suppressed, as will be
discussed later.
80

Two methods for the multiplication of shoots of Pinus pinaster have been
developed by David (30). In the first of these, the axillary bud development
that was described earlier is being made use of. After each new bud has
elongated into a short stem with needles, they are exposed to cytokinin to
induce additional axillary buds. One can thus obtain successive generations of
shoots. In the second method, new apical buds are induced on brachyblasts
(short shoots) by BAP. These elongate and can then be stimulated to form
additional shoots by cytokinin treatment. Furthermore, new brachyblasts are
often formed, each of which may form new apical buds.
Boulay has described a process for the multiplication of shoots of Sequoia
sempervirens (10), and Pseudotsuga menziesii (11) similarly based on the
principle of a succession of axillary buds.

3.2. Adventitious bud formation

This type of morphogenesis is manifested by cells that have acquired a new
function while inside an organ; i.e. they returned to a primary meristematic
state (dedifferentiation). In 1948, La Rue (50) cultured female gametophytes
of Zamia floridana, and obtained adventitious buds in 1% of the cultures.
Since then, studies of shoot formation have progressed considerably, in
particular during the last few years.
After having successively discussed studies dealing with bud induction on
embryos excised from unripe seeds, or on cotyledons, hypocotyl sections, and
needles, we will now consider some general ideas regarding these subjects.
3.2.1. Shoot formation on embryos and cotyledons. Sommer and Brown (66, 67)
cultured embryos of Pinus palustris, that had been removed from seeds soaked
for 36-40 h, and noted bud formation on the cotyledons. The culture medium
contained 1 to 5 mg 1- 1 BAP and 2 mg 1- 1 NAA. The buds, all developing
from cells near the surface (epidermal or subepidermal cells), had to be
transfered to a cytokinin-free medium to avoid degeneration.
However, Sommer (65) observed, that i f Pseudotsuga menziesii embryos were
cultured, a very low dose of BAP induced shoots of a more normal structure.
Therefore, they recommended to use BAP at 0.1 or 1 ppm, with or without 0.01
ppm NAA.
The hormonal requirements varied between species, as was demonstrated by
Coleman and Thorpe (27) in cultures of cotyledons of the gymnosperms Pinus
contorta, Thuja plicata, Cupressus arizonica, and Sequoia gigantea.
 81

On the other hand, Reilly and Brown (62) found that the composition of the
mineral solution did not play a fundamental role in shoot formation on embryos
of Pinus radiata.
Cotyledons of Tsuga heterophylla, cultured by Cheng (24), produced buds if
treated with lAA, IBA and BAP (2.5 to 5 ~mol).

Winton and Verhagen (77) cultured embryos of Pseudotsuga menziesii and

obtained adventitious buds near the apex of cotyledons treated with BAP (0.05
mg 1-1). At a higher concentration (0.1 mg 1-1) the bud formation was
stimulated. Subsequently, only very low concentrations of BAP allowed further
growth. Similarly, Reilly and Washer (63) found bud development at the apex of
cotyledons of embryos of Pinus radiata in culture.
Shoot formation (Fig. 7) was induced on cotyledons of Pinus pinaster (31)
cultured with BAP (10- 5 mol). This cytokinin was more effective than iP or
kinetin in inducing buds on cotyledons of Pseudotsuga menziesii (25) and
embryos or cotyledon sections of Pinus contorta and Picea sitchensis (76).
However, kinetin, although inducing fewer buds, was less inhibitory for the
later stages of development.
Chalupa (19) obtained shoot formation in 65% of Pseudotsuga menziesii
coteyledon sections cultured with BAP (5 mg 1-1) and NAA (0 . 01 mg 1-1).
Wochok and Abo El-Nil (79), and Abo El-Nil and Wochok (1), working with the
same species, noted that on a similar culture medium the organ-forming capacity
of explants diminished with age of the parent seedlings. Organs from plants
only 2-4 weeks old were more responsive than those from more mature plants.
There was also considerable variability between plants from different seeds.
Therefore, they concluded that the shoot forming potential was genetically
determined. The number of chromosomes remained stable during organogenesis.
Maximum adventive bud formation on embryos of Picea abies occurred after a 4
to 5 week treatment with 5 x 10- 6mol BAP or 2-iP. The buds became properly
organized only after transfer of the explants to a hormone-free medium.
Gibberellic acid had no effect. Shoot formation took place in the outermost 5
to 10 layers of cells (71).
Cheah and Cheng (22) followed shoot development by histological methods and
observed that after 4 days in culture, pockets of subepidermal cells started to
divide, and meristemoids were formed. Bud primordia appeared near the surface
of the cotyledons after 20 days in culture, and shoots developed. The struc-
ture of the cotyledons remained largely unchanged because the cell prolifera-
tion was strictly localized. On the other hand, if the medium contained
82

5 II IDol BAP and NAA, or only auxin, many cells divided, a callus was formed,
and the original structure of the explant dissappeared completely.
The site of shoot formation depended on the positioning of the explant on
the nutrient. For example, David (30) and Isemukali (41) observed that if
cotyledons of Pinus pinaster were placed vertical, cell division occurred in a
subterminal position. If the explants were placed horizontal (Fig. 7), all of
the surface became covered with buds. If a nutrient rich in ammonium (10.3
mEq 1- 1 instead of 2.6 mEq 1- 1 ) is used, the ammonium acted as a deter-
minant in shoot formation. It appears that this level of ammonium favors cell
division and creates the conditions required before cytokinin can induce
shoots. The mitotic effect of ammonium was already shown earlier by David
(29). Rancillac (60), culturing embryos of Pinus pinaster, confirmed the
superiority of BAP over kinetin in shoot formation (Fig. 8).
Shoot formation in very young plants (a few days old) and on cotyledons
(Fig. 9) of Pinus sylvestris have been studied by Tranvan and Thomas (69, and
in press). They observed that morphogentic activity does not occur till after
the metabolism of the embryo is activated (after imbibition). The optimal BAP
concentration for shoot formation on the cotyledons ranged from 5 x 10- 6
mol to 10- 4 mol, depending on the age of the cotyledons (excised from
plants 2-10 days old). For the development stage for which 5 x 10- 5 mol is
optimal, a minimum of 3 days is required for shoot induction, the optimum being
6 days. If the cytokinin is applied longer, abnormal structures will develop.
The buds are generally formed in the subterminal part of the cotyledons from
subepidermal cells close to procambial cells that will become involved in
linking the buds to the vascular system of the cotyledons. The authors conclu-
ded that shoot formation did not depend on small, specialized cell groups.
The first indications of the biochemical mechanisms involved in shoot
formation were presented by Hasegawa et al. (40), who found that cytokinin
stimulated the synthesis of low molecular weight (16,000 to 20,000 daltons)
proteins during the first days of application.
Minocha (54) obtained more than 12 buds per Pinus strobus embryo on a medium
with 1 to 2 mg 1- 1 BAP and 1 mg 1- 1 triiodobenzoic acid. These buds were
formed at the apex of the cotyledons.
Finally, buds were induced by Konar and Singh (49) in embryos of Pinus
wallichiana cultured on a Murashige and Skoog medium with BAP and a greatly
reduced amount of ammonium.
 83

Figure 7. Adventitious bud formation along a cotyledon excised from Pinus

pinaster (David).
Figure 8. Formation of buds (B) close to the nutrient medium (~) on the
dorsal side of the cotyledons of a Pinus pinaster embryo in the presence of
cytokinin (Rancillac).
Figure 9. Adventitious bud formation at the tip of cotyledons excised from a
few days old seedling of Pinus sylvestris (Tranvan).

3.2.2. Shoot formation along the hypocotyl. Isikawa (42) cultured hypocotyl
sections of Cryptomeria japonica on a nutrient low in organics (meso-inositol
10 mg 1-1, pyridoxine 0.1 mg 1-1, sucrose 20 g 1-1) and obtained buds in
12% of the cultures. Addition of NAA (0.02 mg 1-1) did not stimulate bud
formation, but increased their elongation rate. The addition of vitamins, malt
extract (500 mg 1-1), and abscisic acid (1 mg 1-1), or BAP (10 mg 1-1),
or NAA (0.1 mg 1-1) with BAP (10 mg 1-1) increased bud formation to 25% of
the cultures.
84

Campbell and Durzan ( l S) studied the behavior of 3-to S-mm-long hypocotyl

sections without apical meristems, that were excised from germinating seedlings
of Picea glauca. The medium contained, besides minerals, the vitamins pyri-
doxine, thiamine, ascorbic acid, riboflavin, and panthothenic acid, and various
concentrations of BAP and NAA. Buds were formed in 60 to 70% of the cultures
on a medium with BAP (10- S mol), with or without NAA (10- 7 mol). The buds
arose from the superficial tissues of the explants. Bud formation in Pinus
banksiana explants was less intense under the same experimental conditions.
Subapical sections of Picea abies hypocotyls formed buds on a medium with
BAP (l-S ppm). Kinetin was ineffective in these cultures (17). It was noted
by Thomas et al. (68) that in cultures of the upper part of the hypocotyls,
with the apical bud and cotyledons left attached, of Biota orientalis and other
Cupressaceae, auxins inhibited bud formation on both the hypocotyls and coty-
ledons . Of the three cytokinins tested (BAP, kinetin, and zeatin) , only BAP
(2.2 ~mol) effectively induced shoot formation, independent of the mineral
nutrition of the explants.
Chalupa (19) induced adventitious buds in hypocotyl sections of Picea abies ,
by using BAP (2 mg 1- 1 ) together with IAA (0.01 mg 1- 1 ). After one month
in culture, 79% of the explants had formed buds.
Continuing their studies of bud formation in hypocotyl sections of Biota
orientalis, Vazart et al. (70) observed that morphogenesis was initiated in the
most external layers of the cortical tissues in certain cells that had remained
mitotically active. Finally, Thomas (in press) determined that while the con-
centration of BAP in the medium controlled the number of buds formed per ex-
plant, the concentration of IBA determined the number of explants that would
react to BAP (Fig. 10).
3.2.3. Shoot formation on needles. After 4 weeks in culture, young needles
excised from Pinus radiata plants 10-12 weeks old, developed meristematic
zones. Transfer of the explants to a medium free of hormones and with only
half strength minerals resulted in bud formation. Explants from seven-and-a-
half month old plants only rarely formed buds. Therefore, the capacity to form
shoots diminishes as the tissues become more differentiated (62).
Comparing the behavior of organs excised from plants of different ages,
Coleman and Thorpe (28) found that shoot formation on needles of Thuya plicata
4-10 years old required five times more BAP than shoot formation on cotyledons,
and required an auxin (NAA 10- 7 mol) as well.
 85

Figure 10. Adventive bud formation along a hypocotyl section of Biota

orientalis (Thomas).

Chalupa (19) obtained buds on needles of Pseudotsuga menziesii and Picea

abies 2-3 months old. The exp1ants were first cultured 4-6 weeks in a liquid
medium with BAP (5-10 mg 1-1) and NAA (0.01 mg 1-1), and then transfered
to a hormone-free medium. Buds are formed a few days after the transfer.
Von Arnold and Eriksson (72) induced shoots from needle primordia of Picea
abies, and on needles from shoots collected shortly after budbreak. They
found that the induction period was reduced from 8 weeks to 2 if the buds were
cultured on liquid medium. BAP (10- 5 mol) was more active than 2-iP at the
same concentration. Organogenesis was obtained in the needle primordia of 5-
50-year-old trees, but only if the shoots were excised from dormant trees. Bud
formation was also induced on very young needles (1-3 mm) removed from growing
shoots. The cells exterior to the mesophyll were the originators of the new
shoots. Finally, the morphogenetic activity of the explants varied between
clones.
Cultures of Pinus sylvestris brachyb1asts, wi th one pair of needles (10-15
mm), formed buds at the base of the needles after 3 weeks on a nutrient con-
taining 20 ~mol BAP (8). Shoot formation also occurred on needles (10-15
mm) of Picea abies (Fig. 11) (44). The optimal hormone combination was 50 nmol
NAA and 5 ~mo1 BAP.
David et a1. (in press) excised brachyblasts, with one pair of needles (70
mm), from cuttings of adult trees of Pinus pinaster that had been sprayed with
a cytokinin solution. Adventitious bud formation occurred in 32% of the
86

Figure 11. A shoot developing near the needle base of Picea abies. The
divisions which give rise to the meristemoids from which the shoot buds are
produced, have their origin in the epidermis (Jansson and Bornman).

explants on a medium with BAP. Histological observations showed that morpho-

genesis occurred at the base of the needle from superficial mesophyll cells in
the elongation zone.
From the foregoing it appears that the ability to induce bud formation in
organ sections has improved considerably in recent years. However, it should
be noted that in most cases bud formation was obtained on embryo (Fig. 8),
cotyledon (Figs. 7 and 9), and hypocotyl (Fig. 10) sections, i.e., in material
that was highly juvenile. Success with organs (needles) from older plants is
far less frequent (Fig. 11). However, it should be pointed out, that the value
of the results obtained so far, ultimately depends on our ability to obtain
similar results with tissues from mature trees. In effect, future efforts
should be concentrated on the vegetative propagation of mature trees selected
on the basis of superior phenotype.
If one uses organs from adult trees, results entirely different from those
obtained with juvenile material are obtained; with age morphogenetic activity
is reduced or completely absent. However, the shoot forming potential may
reappear if one uses organs that have undergone little development (needle
primordia and young needles), or if one treats the parent plant, e.g., with
 87

hormone, before excision of the explants. It has also been observed, that the
morphogenetic capacity of cotyledons rapidly diminishes during the development
of the parent. Therefore, with increasing age of the material, increasingly
stronger hormonal stimuli are required to initiate morphogenesis. These
observations show, that, whatever the nature of the explant or age of the
parent plant, it is the degree of differentiation of certain cells in the
cultured organ that determines its shoot forming potential.
Within each species, the ability to form buds varies between clones. It
would be of interest to determine if this ability could be related to specific
characteristics of vigor, thus allowing proper selection at an early stage,
e.g., in controlled pollination experiments.
Because of lack of systematic studies, there are only a few indications of
what minerals are required to stimulate adventitious bud formation in the ex-
plants. However, it appears that the mineral requirements may not be very
specific. On the contrary, the hormonal requirements and mode of application
for shoot formation have been studied in detail. Amongst the various cyto-
kinins, BAP at about 5 x 10- 5 mol is the most effective. An auxin, gener-
ally NAA at a low concentration, is often used together with the cytokinin.
However, since the hormonal dose required to induce morphogenesis varies be-
tween species, type of explant, and stage of development of the explant, opti-
mal concentrations for bud formation have to be established in each case.
Several studies have shown that an excess of cytokinin does not disturb cellu-
lar dedifferentiation, but interferes with the normal later development of the
buds. Hormones generally have to be applied for a few days to initiate bud
formation; subsequent organization occurs on a medium free of hormones. The
cytokinin concentration appears to determine the number of buds per explant,
the auxin concentration the number of explants that form buds.
The buds originate from cell layers that are exterior to the mesophy11 (in
cotyledons and needles) and the cortex (in hypocotyls). Under the influence of
the hormones, cells that are relatively little differentiated return to a
primary meristematic condition, and thus regain the capacity of division and
bud initiation.
The formation of buds from more or less differentiated cells suggests an
important modification of the behavior of the cell. Because the cells pass
through a stage of unorganized division, one should question the stability of
the genome. Only one study (79) has been carried out on that topic and it
indicated that the chromosome numbers were stable. More work in that direction
88

should be done.

3.3. Embryogenesis
In 1965, Konar and Oberoi (48) cultured embryos of Biota orientalis on
various nutrient media and found what they termed embryos, developing on the
cotyledons. However, later studies by Thomas et al. (68) showed, that actually
these structures were adventitious buds.
Norstog (57) produced embryoids on embryos and female gametophytes of Zamia
integrifolia on a Whi tes' mineral medium wi th extra phosphorus and with
glutamine and alanine. Banerjee and Radforth (5) cultured embryos of Pinus
resinosa at various stages of development. A nutrient medium was used, that
was low in minerals, but supplemented with extracts of the female gametophyte
of Ginkgo. In a few cases, embryos developed from the suspensor.
In 1977, Bonga (6) obtained embryo-like structures on needles of buds of
adult Abies balsamea. Before transfer to the culture medium, the explants were
soaked for 15 minutes in a solution containing 1 g 1-1 IBA, N-dimethylamino-
succanimic acid, or 1-phenyl-3-methyl-5-pyrazolone. Embryoids were also formed
on needles of adult Picea glauca (7), but these structures did not develop past
the cotyledonary stage without becoming disorganized.
In conclusion, only a few publications indicate embryo formation, and some of
these claims may have been based on inadequate identification. Only detailed
histological studies or normal subsequent development of the observed structures
will certify that they are embryos.

3.4. Formation of shoots

Although knowledge about the mechanisms of axillary and adventive bud
formation is improving, less is known about the conditions that control the
formation of shoots from shoot apices. Because of the importance of this
question we will discuss successively studies dealing with elongation of shoots
from dormant buds and from adventitious or axillary buds.
3.4.1. Elongation of shoots from dormant buds. Cultures of dormant buds of
adult Pseudotsuga menziesii required either sucrose, fructose, or glucose at a
concentration of 2% (3). Auxin at a concentration of 10- 6 mol, or less,
had no effect on shoot elongation, but high concentrations stimulated callus
formation. Kinetin was ineffective, and gibberellic acid (0.1-10 mg 1-1),
although stimulating growth initially, caused necrosis of the explants after a
few months in culture. However, addition of urea (5 x 10- 4 mol) results in
 89

good shoot elongation.

While determining the optimal nutritional conditions for the elongation of
dormant buds of various gymnosperms (Picea glauca, Picea abies, Abies balsamea,
Pseudotsuga menziesii), Chalupa and Durzan (21) demonstrated a stimulatory
effect of ammonium chloride (2 mmol), sugar (6%), and urea. Auxin, kinetin,
malt extract, and casein hydrolysate were either ineffective or inhibitory.
Boulay et al. (13) compared the behavior in culture of buds of Pseudotsuga
menziesii excised from juvenile and adult trees. Keeping the cultures at OC
for several months restored meristematic activity. Hormone treatments were
ineffective in that respect. Shoots developed much faster from buds of juve-
nile branches than from buds of mature trees.
Similarly, Qlalupa (18) observed that buds excised from Picea abies less
than 2 years old, elongated twice as fast as buds from adult trees, which elon-
gated 3-6 cm in 6-8 months. The addition of IAA (0.01-0.3 mg 1- 1 ), and gib-
berellic acid (0.01-0.2 mg 1- 1 ) stimulated elongation.
Finally, Boulay (11) noted that excision just before bud break provided the
fastest elongating shoots. This suggests, that the bud meristems accumulate
hormones and nutrients shortly before their reactivation in the spring.
3.4.2. Elongation of shoots from adventitious and axillary buds. No syste-
matic studies have been carried out in this area. Therefore, the discussion
will be limited to the culture conditions used by various researchers to main-
tain the apex sufficiently functional to obtain stems with leaves.
Low concentrations of IAA (10-8 -10- 11 mol) stimulated elongation of
axillary buds of Pseudotsuga menziesii (12). To induce elongation, some au-
thors, (19, 71, 79) have transferred explants with newly formed buds, or buds
removed from the explants, to nutrient media free of hormones and often with
only half the normal mineral strength.
Activated charcoal (Figs . 12 and 13) at concentrations between 0.5 and 2%
are often added to the medium (10, 32).
Konar and Singh (49) obtained shoot elongation after transfer of adventi-
tious buds of Pinus wallichiana to a medium with 10% coconut milk.
The number of publications discussing shoot elongation is small and deals
only with dormant shoots. The reported improvements, in particular in inorgan-
ic and organic nitrogen application, have not lead to continued growth of axil-
lary and adventitious buds. In general, it appears that conditions that stimu-
late abundant shoot induction, inhibit continued shoot growth. Transfer to a
medium free of cytokinin or to a medium with auxin at very low concentrations
90

stimulates elongation.

Figure 12 . The influence of activated charcoal on the elongation of Sequoia

sempervirens shoots: at left, on a control medium, at right, on a medium with
2% activated charcoal (Boulay).
Figure 13 . A shoot of Pinus pinaster cultured on a medium with 0.5% activated
charcoal (David).

Detailed studies with gibberellic acid have shown contradictory results in

gymnosperm cultures . Sometimes it stimulated elongation, in other experiments
it was ineffective or toxic. However, Maheshwari et al. (52) obtained shoot
elongation in cultures of Cuscuta chinensis treated with gibber.e llic acid, and
therefore, studies with gibberellic acid in gymnosperm cultures should be con-
tinued.
Often shoots are transplanted to a medium containing only half the strength
of the minerals used to initiate shoot formation. Addition of activated car-
bon, following the technique of Martin et al. (53), stimulates elongation, but
optimal concentrations have not yet been properly determined (Figs. 12 and 13).
Part of the action mechanisms of activated charcoal is based on absorption of
hormones, phenolics, and toxins created during autoclaving (37, 74, 75).
 91

Several authors have indicated that shoot elongation does not occur till
after root formation. This demonstrates the influence of the root system on
the function of the shoot apex, and suggests that early root induction could be
a means to obtain shoot elongation. Furthermore, rooted buds would benefit
from the juvenile influence exerted by the roots. This effect is of consider-
able importance in propagation experiments with material from mature trees, as
will be discussed later.
Finally, it should be pointed out that the elongation rates of shoots in
vitro is always much lower than those in situ. This shows that the culture
medium does not compensate for the absence of the root system, and that culture
conditions are not optimal yet.
It serves no useful purpose to perpetually produce new generations of buds
if these cannot be stimulated to grow into normal shoots, and more research to
solve this problem is necessary. The technique of axillary bud formation per-
mits accumulation of genetically homogenous bud populations, and thus provides
suitable material for future studies.

3.5. Root formation

Only studies dealing with root formation on shoots that developed from dor-
mant buds, axillary buds, or adventitious buds will be discussed.
Chalupa (17) and Sommer (65) occasionally obtained roots in cultures of
Picea abies and Pseudotsuga menziesii, respectively, after IBA or NAA treat-
ments.
Sommer et al. (67) obtained rooting of adventitious buds of Pinus palustris
after transfer of the buds to an auxin-and cytokinin-free, but vitamin-con-
taining medium . Explants that had not rooted after 5-6 weeks were transfered
and cultured for one month on a medium containing IBA (10 mg 1-1), and then
returned to the hormone-free medium. In several of the explants, roots devel-
oped subsequently. About 1% of the shoots that had originated from needles of
Pinus radiata rooted if cultered for 10 days on a medium with 5 mg 1-1 IBA.
Rooting was not improved if substrates other than agar, such as sand, peatmoss,
or vermiculite were used . Therefore, the substrate appears not to be a deter-
minant in root induction (62).
Chalupa (19) induced rooting in shoots of Pseudotsuga menziesii by culturing
them on a medium low in minerals and sucrose (0.5%), and with NAA (0.02-0.1
mg 1-1).
92

Figure 14. Root formation in shoots of Pseudotsuga menziesii (Boulay and

Franclet)
Figure 15. Root formation in shoots of Sequoia sempervirens (Franclet and
Boulay).

Boulay and Franclet (12) obtained roots in shoots (Fig. 14) that developed
from dormant buds excised from Pseudotsuga menziesii plants 6 months, 2, and 4
years old, and from epicotyls from plants 4-6 weeks old. The media used con-
tained one-half or one-third of the mineral concentrations used for shoot
growth. The most effective auxin for rooting was lAA at high concentrations
(10-20 mg 1-1). Rooting is influenced by the age of the parent plant at
the time the buds were excised. The best results were obtained with parent
plants less than 2 years old. When dealing with epicotyl sections, auxins were
not required. The authors also emphasized the importance of the substrate in
root formation and advised that substrates should allow good aeration.
Shoots of Pinus pinaster that had developed from axillary buds were cul-
tured by David and David (31) in a medium with IBA (10- 7 mol), vitamins,
and amino acids. A few of the shoots formed roots.
Root formation was influenced by the origin of the explants that formed
shoots. Coleman and Thorpe (28) noted that in the presence of IBA (5.10- 5
mol) 50% of the shoots that formed on cotyledon explants formed roots,
while in shoots induced on explants of 4- to 10-year-old trees, only 11% did
so. They also showed that the physiological state of the tissues not only in-
fluences bud induction, but also the subsequent behavior of the newly formed
buds.
Cheng and Voqui (26) obtained rooting in 80% of the adventitious shoots that
developed from cotyledon sections of Pseudotsuga menziesii. The young shoots
were cultured on an agar medium containing minerals, a low concentration of
sucrose (0.5%), and NAA (0.25 ~mol). They noted that at a temperature of
19C satisfactory rooting occurred and that the plant lets developed normally.
If the temperature was raised to 24C, callus was formed and the plantlets
showed abnormal growth. They concluded, that depending on the temperature, the
auxin affected different types of cells.
Webb and Street (76) demonstrated that rooting of adventitious shoots of
Pinus contorta and Picea sitchensis was influenced by the type of cytokinin
that had been used for induction of the shoots. If 2-iP or kinetin had been
used for shoot induction, more roots were formed than if BAP had been used.
They also noted that cytokinins with a strong shoot inducing capacity inhibited
both rooting and stem elongation of the shoots.
Rooting of adventitious shoots of Pinus radiata was obtained by Reilly and
Washer (63) on a medium low in minerals, supplemented with NAA (0.5 ppm) and
IBA (2 ppm). Root elongation was subsequently stimulated by a 2-3 week soak in
distilled water.
Chalupa (18) found that shoots that had developed from dormant buds of Picea
abies rooted easier if the buds had been excised from trees less than 2 years
old, than if they had been excised from older trees.
Root formation (Fig. 16) was induced in shoots of Pinus pinaster after
soaking for 24 h in an IBA solution followed by transfer to a well aerated,
sterile mixture of perlite and peatmoss (32). Rooting occurred in 80% of the
shoots from axillary buds of 20-day-old Pinus pinaster plants. Only 50% of the
shoots from the apex of brachyblasts of 2- to 3-year-old trees rooted.
Using the same species, Rancillac (60) found that the auxin concentration
affected the quality of the roots. NAA, at 10- 6 mol, induced many roots at
the base of the shoots, but also callus, which often prevented the establish-
ment of vascular connections between roots and shoots. On the other hand, a
concentration of 10- 7 mol resulted in thin, normal roots, and no callus at
the base of the shoot. A temperature of 20C resulted in better root growth
than 25C. A nutrient, low in minerals, was best for root growth. Transfer to
94

Figure 16. A root system developed from a shoot of Pinus pinaster on an

aerated substrate. Note the presence of mycorrhizogenic roots (David).

a greenhouse was possible after the roots had grown for 2-3 months on the
sterile medium.
Shoots from dormant buds of Pseudotsuga menziesii were rooted by Boulay
(11), but rooting occurred only if the buds were taken from trees less than
4-years old.
Minocha (54) found that some adventitious buds of Pinus strobus, formed 1
or 2 roots if treated with 0.01-2 mg 1-1 IBA and subsequently cultured on
agar, or on a solidified mineral solution. The author concluded that the low
rooting percentages were the result of an excess of endogenous hormones. High
concentrations of sucrose (3-6%) favored rooting.
Bornman and Jansson (8) obtained rooting in 35% of short shoots of Pinus
sylvestris treated with coumarin (10 ~mol).

To improve rooting, some investigators have tried rooting under non-sterile

conditions, which has the advantage of simplifying the procedures and allowing
the use of a variety of solid substrates. Chalupa (18) thus obtained rooting
of Picea abies and Pseudotsuga menziesii shoots on perlite. The base of the
shoot was wetted and dipped in a powder containing NAA (500 mg 1-1), nico-
tinic acid (500 mg 1-1), and thiamine (10 mg 1-1). The shoots were mist
sprayed twice a day with a mineral solution, and kept under high atmospheric
humidity.
Shoots of Sequoia sempervirens were rooted by Boulay (10), following a
similar procedure. He soaked the base of the shoots for 24 h in an IBA
 95

-fungicide solution. The shoots were then planted on a mixture of perlite and
vermiculite (4:1) and maintained in a damp atmosphere. About one month later,
up to 80% of the shoots had rooted. The results varied, depending on the vigor
of the shoots at the time of root induction.
Adventitious root formation was also studied by Faye et al. (in press).
They noted formation of two types of roots. In one case, roots elongated from
pointed meristems, in the other, short dichotomously branching roots developed
from rounded meristems. The short roots were similar to roots with mycorrhiza
found on field grown trees. Therefore, the formation of roots resembling those
with mycorrhiza does not depend on the symbiotic fungus.
Root formation in gymnosperms (Figs. 14, 15, and 16) has been studied by
only a few investigators. Root formation has generally only been possible if
the shoots originated from embryos or plants from a few days to a few years
old. Many investigators have observed spontaneous rooting without the aid of
exogenous growth hormones, but the rooting percentages were generally low
(about 1%).
Generally, NAA and/or IBA are used to induce root meristems. It appears
that the duration of application (from 24 h to continuous) required is inver-
sely proportional to the concentration. As in shoot formation, the quality of
the adventitious root system depends on the intensity of the induction stimu-
lus. A strong activation results in many primordia, but in some species the
roots formed from these may not be functional.
Generally, a lowering of the mineral concentration to one-half or one-third
of the concentration used for shoot formation, favored root development. Some
investigators found that cytokinin combinations that favored shoot induction,
subsequently inhibited the formation of new roots. The cytokinin effect was
residual, i.e., it was still noticeable several weeks after removal of the
cytokinin from the medium. Furthermore, the rooting ability like the shoot
forming and stem elongating ability was reduced with increasing age of the
parent plant from which the explants were taken.
Although auxins are generally used for root induction, Smith and Thorpe (64)
have shown that in addition some amino acids and phenylpropanoids were involved
in the formation of root primordia in hypocotyls of Pinus radiata. Haissig
(39) postulated that root formation occurs only if auxins, certain phenolics,
specific oxidases, and activators of these enzymes are present together at a
non-specific site. These substances act synergistically in the induction of
root primordia.
96

4. REGENERATION FROM EXPLANTS FROM MATURE PLANTS; REJUVENATION

Most investigators have tried to vegetatively propagate selected mature
trees by using dormant buds in vitro (6, 12, 13, 18, 21, 72, and Cornu, Per-
sonal Communication). With the exception of one study (18), none have yet re-
ported regeneration from mature plants. In general it appears to be difficult
to obtain normal shoot elonga tion and rooting of the shoots.
Because of the lack of morphogenesis in explants taken from mature trees,
some investigators have used parts of adult trees that were juvenile. For
example, Boulay (10) propagated Sequoia sempervirens more than 50 years old, by
using explants taken from stump sprouts. He noted a lower rate of propagation
from sprouts 2 years old, than from sprouts in their first season.
Because only a few gymnosperms produce stump sprouts, Franclet (35, in
press) tried several treatments to induce at least partial rejuvenation of
material before its in vitro culture. Thus, the method of repeated grafting of
apices on juvenile plants was established, which has resulted in rejuvenation
of clones of Pseudotsuga menziesii aged 15 and 75 years old. Explants taken
after four or. five successive graftings have shown a juvenile behavior in
vitro, comparable to that in autografts. Therefore manipulation of vegetative
material from mature trees, before the in vitro phase, can restore the morpho-
genetic capacity of its cells. Subsequent in vitro culture could lead to more
normal development of the propagules, especially if during culture exposure to
cytokinin is followed by exposure to activated charcoal. For example, if one
compare~ shoots of Sequoia sempervirens that have passed through four succes-
sive in vitro cycles (one cycle = one period of exposure to cytokinin followed
by one of exposure to activated charcoal) with traditional cuttings, one will
notice a more rapid rooting and reduced plagiotropism in the in vitro material.
This indicates a rejuvenation of the material (Fig. 17).
Finally, Franclet et al. (36) have obtained regeneration from Pinus pinaster
trees 11 years old. In vitro cultures were established of brachyblasts that
were excised from current shoots from rooted cuttings that had been intensively
treated wi th fertilizer. These treatments had a rejuvenating effect, and
plants with a morphology comparable to that of young seedlings were regenerated
(Fig. 18). It was further observed, that the ultimate behavior is strongly in-
fluenced by the environmental conditions applied after the in ~:tro phase. A
rapid transfer to soil leads to reduced vigor, the formation of long needles,
bud dormancy, and a return to the physiological state of the mature tree. On
the other hand, if the plants are maintained, after the in vitro phase, under
 97

Figure 17. Propagules of Sequoia sempervirens; left, rooted cuttings remained

plagiotropic; right, propagules derived from in vitro cultures have become ort-
hotropic (Boulay and Franclet). -- -----
Figure 18. Rejuvenation of Pinus pinaster; the four specimens belong to one
clone. a) Seedling derived from a germinating seed of an 11-year-old tree.
b) Specimen regenerated in vitro from an axillary bud from a plant a few days
old. c) Rooted cutting from the 11-year-old tree. d) Specimen regenerated in
vitro from a brachyblast with one pair of needles. The brachyblast was ex---
cised from a cutting of the 11-year-old tree (see c). Note, that the needles
are juvenile. This indicates, that the level of juvenility is comparable to
that of a seedling (see a), or to that of a plant derived from seedling tissues
in vitro (see b) (Franclet, David, and Boulay).
98

optimal nutritional and rooting conditions, it is possible to temporarily

prolong the juvenile phase.

5. ESTABLISHMENT OF PROPAGULES IN SOIL

Sommer and Brown (66) and Sommer et al. (67) developed new plants from
embryo tissues of Pinus palustris and established these in soil.
Adventitious shoots of Picea glauca were rooted by Campbell and Durzan (16).
They subsequently placed these rooted shoots on a sand and sphagnum mixture,
where they rapidly developed into seedlings. These seedlings were watered
weekly with a weak mineral solution.
Plants that developed from young needles of Pinus radiata continued growing
after transfer to a sand-peat mix (62) and normal plants of Thuja plicata were
obtained on a peat-vermiculite (1:1) mixture in a humid atmosphere (28).
To improve root growth on rooted shoots, Reilly and Washer (63) soaked the
shoots for 2-3 weeks in water, before transferring them to soil. This assured
better growth of the plants after the transfer. Ultimately mycorrhiza were
formed on the roots.
To reduce the mortality of plants after their transfer from in vitro to
soil, Cheng (24) rooted Tsuga heterophylla shoots on a mixture of vermiculite-
peat under non-sterile conditions. This exposed the plants to soil microor-
ganisms, which probably explains their improved growth.
New plants of Picea abies (18), Pseudotsuga menziesii (11, 18, 19), and
Pinus radiata (2) were obtained from various explants from very young specimens
and established in soil. Pinus pinaster (Figs. 19 and 20) was regenerated from
axillary buds (30, 31, 60, 61), from adventitious buds, and from apical buds of
brachyblasts (30, 31) and subsequently grown in soil.
Finally, Bornman and Jansson (8) similarly obtained new plants of Pinus
sylvestris after growth in a greenhouse.

6. CONCLUSIONS
In summary, regeneration of plants has been obtained with several species.
In most cases the explants were embryos, and organs, or organ sections from
specimens up to a few years old. The applications of such techniques are
numerous. Of particular interest are studies of genotype-environment inter-
actions, testing of clones for cold and pest resistance, and the rapid multi-
plication of controlled crosses.
 99

Figure 19. A Pinus pinaster plant, that originated from an axillary bud of a
very young plant, growing in a nursery bed (David).
Figure 20. A Pinus pinaster plant, that originated from an adventitious bud on
a cotyledon, growing in a nursery bed (David).

However, a tree does not express its full genetic potential till it reaches
the mature stage. During this maturation process several physiological changes
take place in the tree that affect the in vitro behavior of the explants taken
from the tree at various times. This effect has been demonstrated in the
ability to form adventitious and axillary buds, in the rate of shoot elonga-
tion, and rooting. Because of the decreased morphogenetic ability of mature
material it is generally not possible to apply the techniques developed for
juvenile material to mature plants.
Present research is showing that treatment of the plants before excision of
the explants and in vitro culture, as well as manipulation of the environment
after regeneration, will occasionally result in the reappearance of juvenile
behavior. These results raise the expectation, that soon the systematic multi-
plication of selected mature trees will become possible.
Vegetative propagation produces uniform copies, and thus provides a tool for
the propagation of genetically improved specimens. However, the classical
100

procedures have two major problems; low efficiency under nursery conditions,
and difficulties in propagating mature trees. Because tissue culture research
has lead to new, efficient methods of vegetative propagation of several angio-
sperms, a similar approach has been attempted with gymnosperms, but so far with
only limited success.
In comparison with the classical propagation techniques, the experimenter
has greater manipulative control over cells and tissues with in vitro tech-
niques. Furthermore, the classical methods have always suffered from seasonal
constraints. In vitro techniques may remove some of these, and thus improve
the annual propagation rates.
With many gymnosperm species, in vitro propagation presently is possible
only if juvenile material is used. This often provides axillary or adventi-
tious buds, which, if they properly elongate and form roots, will grow into
viable new plants. The ability to form buds, elongated shoots, and roots de-
pends on the species; for example, in some cases shoot elongation is exces-
sively slow, in others rooting is difficult. These problems indicate, that the
optimal conditions for development during each of these phases have not yet
been defined, and that further research is required. Following the in vitro
culture phase it is essential to transfer the new plants to a proper substrate
and to maintain temporarily a high atmospheric humidity.
Methods similar to those used for the propagation of juvenile material could
eventually lead to in vitro vegetative reproduction of mature trees, selected
for their superior phenotype. However, to date, the results with mature trees
have generally been only partially successful. The difficulties encountered
are largely caused by physiological changes that occur during maturation of the
trees. Presumably in vitro culture removes most internal correlations, but
this is not sufficient to rejuvenate the cells in explants from mature trees.
Although some treatments applied during the in vitro phase may initiate par-
tial rejuvenation, the condition of the donor plant at the time of explant
excision is also important. Therefore, research regarding the proper condi-
tioning of the donor plant should be intensified.
Foreseeing the possibility of somatic hybridization, and genetic transforma-
tion of cells by incorporation of organelles, or molecules (see Chapter 12),it
becomes even more essential to master the morphogenetic control mechanisms of
the cells. When techniques are developed to raise new plants from such genet-
ically modified cells, it should be established whether or not the character-
istics acquired through genetic manipulations, are stable.
 101

The recent success in regenerating gymnosperms from juvenile, and sometimes

from mature material in vitro should encourage researchers to continue their
search for methods to overcome the difficulties that are presently hindering
the systematic improvement of the various forest tree species.

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hypocotyl of Crypt~a japonica. Bot Mag 87 : 73-77
43. JACQUIOT C 1955 Sur Ie role des correlations d'inhibition dans les pheno-
menes d'organogenese observes chez Ie tissu cambial, cultive in vitro, de
certains arbres. Incidence sur les problemes du bouturage. CR~cad Sc 241
1064-1066
44. JANSSON E, C BORNMAN 1980 In vitro phyllomorphic regeneration of shoot buds
and shoots in Picea abies.-Physiol Plant 49 : 105-111
45. JOHNSON MA, JA CARLSON 1977 Attempts to induce embryogenesis in conifer
suspension culture: Biochemical aspects. Tappi, Inst. Paper chemistry,
Appleton, Wisconsin, 25-29
46. KADKADE PG, H JOPSON 1978 Influence of light quality on organogenesis from
the embryo-derived callus of Douglas fir (Pseudotsuga menziesii). Plant Sci
Lett 13 : 67-73
47. KONAR RN 1975 In vitro studies of Pinus. II. The growth and morphogenesis
of cell cultures from Pinus gerard~ Phytomorphology 25 : 55-59
48. KONAR RN, YP OBEROI 1965 In vitro development of embryoids on the cotyle-
dons of Biota orientalis.-Phytomorphology 15 : 137-140
49. KONAR RN~SINGH 1980 Induction of shoot buds from tissue cultures of
Pinus wa1lichiana. Z Pflanzenphysiol 99 : 173-177
50. LA RUE CD 1948 Regeneration in the megagametophyte of Zamia floridana.
Bull Torrey Bot Club 75 : 597-603 -----
51. LUTZ A 1969 Etude des aptitudes morphogenetiques des cultures de tissue.
Analyse par 1a methode des clones d'origine unicellulaire. Rev Gen Bot 76
309-359
52. MAHESHWARI R, C SHAILINI, K VELUTHAMBI, S MAHADEVAN 1980 Interaction of
gibberellic acid and indol-3-acetic acid in the growth of excised cuscuta
shoot tips in vitro. Plant Physio1 65 : 186-192
53. MARTIN C, M-cARRE, G DUC 1979 Note sur les cultures de tissus de feverole
(Vicia faba L.); bouturage, culture de cals, culture de meristemes. Ann
Amelior Plantes 29 : 277-287
54. MINOCHA SC 1980 Callus and adventitious shoot formation in excised embryos
of . white pine (Pinus strobus). Can J Bot 58 : 366-370
55. NITSCH JP, C NITSCH 1970 Obtention de p1antes haploides a partir de pollen.
Bull Soc Bot Fr 117 : 339-360
56. NORREL B, JP NITSCH 1968 La formation d'''embryons vegetatifs" chez Daucus
carota L. Bull Soc Bot Fr 115 : 501-514 ------
57. NORSTOG K 1965 Induction of apogamy in megagametophytes of Zamia integri-
folia. Am J Bot 52 : 993-999 -----
58. NORSTOG K, E RHAMSTINE 1967 Isolation and culture of haploid and diploid
cycad tissues. Phytomorpho10gy 17 : 374-381
59. RAMAWAT KG, HC ARYA 1976 Growth and morphogenesis in callus cultures of
Ephedra gerardiana. Phytomorphology. 26 : 395-403
60. RANCILLAC M 1979 Mise au point d'une methode de multiplication vegetative
in vitro du Pin maritime (Pinus pinaster Sol.) pour la constitution de
clones a partir de semences.Etudes et recherches, AFOCEL, Domaine de
l'Etan~on, 77370 Nangis, France, 12 : 41-48
61. RANCILLAC M 1981 Perspectiv.es d'application des cultures d'organes in vitro
a la multiplication vegetative du Pin maritime, Pinus pinaster Sol.~nn Sci
For 38 : 55-69
62. REILLY K, CL BROWN 1976 In vitro studies of bud and shoot formation in
Pinus radiata and Pseudotsuga menziesii. Ga For Res Pap 86 : 1-9
63. REILLY K, J WASHER 1977 Vegetative propagation of radiata pine by tissue
culture. Plant let formation from embryonic tissue. NZ For Sci 7 : 199
104

64. SMITH DR, TA Thorpe 1977 Root initiation in cuttings of Pinus radiata seed-
lings : effects of aromatic amino acids and simple phenyl propanoids. Bot
Gaz 138 : 434-437
65. SOMMER HE 1975 Differentiation of adventitious buds on Douglas fir embryos
in vitro. Proc Int Plant Prop Soc 25 : 125-127
66. SOMMER HE, CL BROWN 1974 Plantlet formation in pine tissue cultures. Am J
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67. SOMMER HE, CL BROWN, PP Kormanik 1975 Differentiation of plantlets in long-
leaf pine (Pinus palustris Mill.) tissue cultured in vitro. Bot Gaz 136 :
196-200 -- - --
68. THOMAS MJ, E DUHOUX, J VAZART 1977 In vitro organ initiation in tissue cul-
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Lett 8 : 395-400
69. TRANVAN H 1979 In vitro adventitious bud formation on isolated seedlings of
Pinus silvestriS-L. BioI Plant 21 : 230-233
70. VAZART J, M DA CONCEICAO, MJ THOMAS 1979 Structure anatomique et cytolo-
gique de l'hypocotyle du Biota orientalis L. au stade de l'etalement des
cotyledons. Rev Cytol BioI Veget 2 : 83-96
71. VON ARNOLD S, T ERIKSSON 1978 Induction of adventitious buds on embryos of
Norway spruce grown in vitro. Physiol Plant 44 : 283-287
72. VON ARNOLD S, T ERIKSSON 1979 Induction of adventitious buds on buds of
Norway spruce (Picea abies) grown in vitro. Physiol Plant 45 : 29-34
73. VON ARNOLD S, T ERIKSSON 1979 Bud induction on isolated needles of Norway
spruce (Picea abies) grown in vitro. Plant Sci Lett 15 : 363-372
74. WEATHERHEAD MA~URDON, GC-HENSHAW 1978 Some effects of activated char-
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141-147
75. WEATHERHEAD MA, J BURDON, GG HENSHAW 1979 Effects of activated charcoal as
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399-405
76. WEBB KJ, HE STREET 1977 Morphogenesis in vitro of Pinus and Picea. Acta
Hortic 78 : 259-269 - -- -- --
77. WINTON LL, SA VERHAGEN 1977 Shoots from Douglas fir cultures. Can J Bot 55
: 1246-1250
78. WINTON LL, S VERHAGEN 1977 Embryoids in suspension cultures of Douglas fir
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79. WOCHOK ZS, M ABO EI-NIL 1977 Conifer tissue culture. Proc Int Plant Prop
Soc 27 : 131-135
 !O5

APPENDIX

In Vitro Cultures of Gymnosperms

Species Type of explant Type of development References

CYCADALES

Cycas circinalis Megagametophyte Embryogenesis 58

callus

Zamia floridana Megagametophyte Shoo t formation 50

Zamia integrifolia Proembryo call us Embryogenesis 58

Megagametophyte Embryogenesis 57
Embryo Embryogenesis 57

GNETALES

Ephedra gerardiana Subcultured Embryogenesis,

tissues shoot and root
formation 59

CONIFERALES

Pinaceae

Abies balsamea Shoots Axillary bud

formation,
embryogenesis 6

Buds Shoot formation 21

Laris x eurolepis Stem segment Axillary bud Personal

formation Cornrnunic.

Picea abies Cell suspension Embryogenesis 20

Embryo call us Shoot formation 19
Embryo Shoot formation 71
Hypocotyl Shoot formation 17,19
Needle Shoot formation 19,45,72,73
Bud Shoot formation 18,21,71,72
Shoot Root formation 15,17,18
Plant in soil 18,19

Hypocotyl Shoot formation 15

Needle Embryogenesis 7
Bud Shoot formation 21
Shoot Root formation,
plant in soil 16
106

Continued

Species Type of explant Type of development References

Picea mariana Bud Shoot formation 21

Picea sitchensis Embryo callus Shoot formation 76

Embryo Shoot formation 76
Cotyledon Shoot formation 76
Shoot Root formation 76

Pinus banksiana Hypocotyl Shoot formation 15

Pinus contorta Cotyledon Shoot formation 27,76

Embryo Shoot formation 76
Shoot Root formation 76

Pinus gerardiana Subcultured tissues Shoot and root

formation 47

Pinus palustris Embryo Shoot formation 66,67

Shoot Root formation,
plant in soil 67

Pinus pinaster Apex with Axillary bud

cotyledons formation 30,32,41,60
Brachyblast with Apical bud
needles formation 30
Cotyledon Shoot formation 30,31,41
Embryo Shoot formation 60,61
Bud Shoot formation 32
Shoot Root formation, 30,31,32,36
plant in soil 60,61

Pinus radiata Embryo callus and

juvenile needles Shoot formation 62
Cotyledon call us Shoot formation 63
Embryo Shoot formation 62
Cotyledon Shoot formation 63
Needles Shoot formation 62
Shoot Root formation,
plant in soil 2,62,63,64

Pinus resinosa Embryo Embryogenesis 5

Pinus sylvestris Cotyledon Shoot formation 69

Brachyblast with Shoot formation 8
needles
Shoot Root formation,
plant in soil 8
 107

Continued

Species Type of explant Type of development References

Pinus strobus Embryo callus No organogenesis 54

Embryo Shoot formation 54
Shoot Root formation 54

Pinus taeda Cell suspension Embryogenesis 78

Pinus wallichiana Embryo Shoot formation 49

Embryo callus Shoot formation 49
Bud Shoot formation 49

Pseudotsuga Subcultured tissues Shoot formation 23

menziesii Embryo callus Shoot formation 19,46
Cotyledon callus Shoot formation 75
Cell suspension Embryogenesis 34,45,78
Bud Axillary bud
formation 9,12
Embryo Shoot formation 65
Cotyledon Shoot formation 1,19,22,25
40,77,79
Needles Shoot formation 19
Bud Shoot formation 11,12,13,18,
19,21,79
Shoot Root formation 11,12,15,19,
26,65
Plant in soil 11 ,18 ,19

Pseudotsuga Bud Shoot formation 3

taxi folia

Tsuga heterophylla Cotyledon Shoot formation 24

Shoot Root formation,
plant in soil 24

Taxodiaceae

Cryptomeria japonica Hypocotyl Shoot formation 42

Sequoia gigantea Cotyledon Shoot formation 27

Sequoia Subcultured tissue Shoo t formation 4

sempervirens Stem segment Axillary bud
formation 10
Bud Shoot formation 10
Shoot Root formation,
plant in soil 10
108

Continued

Species Type of explant Type of development References

Cupressaceae

Biota oriental is Hypocotyl callus Shoo t and roo t

formation 68
Hypocotyl, embryo Shoot formation 68,70
Embryo Embryogenesis 48

Cupressus arizonica Cotyledon Shoot formation 27

Thuja plic a ~,,: Cotyledon Shoot formation 27

Leaves Shoot formation 28
Shoot Roo t formation,
plant in soil 28
 109

5. VEGETATIVE PROPAGATION OF DICOTYLEDONOUS TREES

C . L. BROvm AND H. E . SOX.'1ER

1. INTRODUCTION
Vegetative propagation of woody dicots possessing one
or ~ore desirable genetic traits has been practiced by man
for centuries. Numerous species of ornamental, fruit, or
nut trees among diverse genera have been cloned successfully
by rooted cuttings, layering, or budding and grafting depend-
ing upon species response, utility and needs (48, 29).
Asexual propagation has been used infrequently in commercial
forest production because most forest trees are difficult to
root past their juvenile stage and the millions of plants
needed in reforestation programs each year have been more
easily produced from seed. with ~nprecendented progress in
tissue culture technology during the past decade, coupled
with the continued spiraling demand for cellulose throughout
the world, one can predict with certainty that asexually
produced trees will comprise a significant portion of forest
planting stock in the future.

2. USE OF CONVENTIONAL HETHODS OF VEGETATIVE PROPAGATION

IN PRODUCTION FORESTRY
2.1. Past practices and utility
One of the major difficulties encountered in vegetative
propagation of most forest trees is the inverse relationship
existing between aging and ease of rooting stem cuttings
(see Chapter 13). Nevertheless, some economically important
trees can be propagated with little difficulty using dormant
hardwood cuttings planted directly into field plots. Various
species of Salix and non-aspen Populus have been regenerated
for quite some time using stem cuttings of selected strains
110

or hybrids, which are multiplied in nursery beds or clone

banks under intensive culture. Once established the vigorous
shoots may be cut down after one or two years and allowed to
resprout from the stumps. Thereafter dormant sprouts may be
harvested annually and cut into sections of varying lengths
(frequently 30-50 cm) and planted directly in the field (65,
89). Today this technology has been expanded and improved,
and, depending on the cultivars of Salix or Populus and the
soil types encountered in reforestation is being used for
commercial production. An excellent detailed account of
nursery production and explanting of propagules of various
ages has been recently published under the auspices of the
International Poplar Commission of FAa (23).
Many other reports are found in the literature concerning
methods and techniques of rooting stern cuttings of various
economically important hardwoods in both temperate and
tropical areas around the world. Most of these studies have
been conducted with auxin treated (e.g., basal dips) stern
cuttings placed in mist beds in a lathhouse or greenhouse
under favorable temperature and photoperiodic regimes.
Although numerous species have been successfully propagated
in this way, rooting responses in most trials have not been
consistently high enough to warrant mass production of
commercial planting stock. So far the utility of conventional
propagation has been largely restricted to establishment of
clonal banks of selected phenotypes, or in studying clonal
responses of selected parent trees on different sites. The
literature covering research in this field is largely empirical
and need not be discussed here in detail. Among temperate
zone trees variable rooting success has been noted in species
of Populus (aspens), Acer, Quercus, Juglands, Liquidambar,
Liriodendron, Prunus, Platanus, Ulmus, Fraxinus, Nyssa,
Cornus, Betula, and Castanea (6, 24, 30, 42, 51, 77, 79).
In tropical or sub-tropical areas several commercially im-
portant trees have been propagated to some extent in genera
of Eucalyptus, Bombax, Dalbergia, Tectona, Morus, Aesculus,
Platanus, Toona, Lagerstroemia, Banbinia, Ailanthus, Boswellia,
 III

Broussonetia, Casurina, Cinnamornum, Gmelina, Ficus, Garuga,

Hymenodictyon, Lannea, Michelia, Shorea, and Terminalia (5,
20) .
As more and more demands are made annually on forest
productivity accompanied by demonstration of enhanced yields
with selected and tested clones, many of the more conventional
means of propagation, or improved modifications, thereof,
will likely become economically feasible and attractive in
various reforestation schemes. certainly, in many sub-
tropical and tropical areas of the world various conventional
propagation procedures along with improved containerization
practices hold much promise in high yielding, short-rotation
forest crops (see Chapter 3).
As is always the case, however, one usually encounters
genotypic variation in the rooting response of selected fast
growing phenotypes even among so called "moderately easy-to-
root species". Unfortunately, this genotypic variability
also exists when one uses tissue culture techniques to
obtain organogenesis (bud and root formation), and this
problem still presents real difficulty in developing practical
systems of mass propagation of selected high yielding strains
of forest trees. Some progress is currently being made to
circumvent this difficulty as discussed in Section 5.1.

2.2. Modified approaches and applications

Recognizing that rooting response rapidly declines with
tree age, one may use several techniques to obtain physiologi-
cally younger material or so called "rejuvenated material"
for establishing clone banks to enhance the rooting response
(see Chapter 13). For example, partial girdling the main
trunk just above the root collar frequently releases suppressed
buds to form stump sprouts which may revert to a more juvenile
condition and be rooted with a fair degree of success (50).
Likewise, partial stem girdles, placed along the lower main
bole results in the release of suppressed buds to form
epicormic branches which can frequently be rooted with
success in mist beds (42). Garner and Hatcher (26) and
112

Libby (45) have used the technique of severe pruning or

hedging to arrest the maturation phase for propagating
otherwise difficult to root woody material. There are also
several indications that some of the difficulty associated
with aging can be overcome by grafting older scion material
onto young root stock to obtain rejuvenation (16). It may
be necessary to repeat the grafting cycle several times in
succession prior to taking cuttings from the grafted scions
to obtain adequate rooting. Once clones are established
additional cuttings may be taken as in hedging to maintain
the rejuvenated phase (52). This technique appears to work
for certain species of Eucalypts (see Chapter 6).
Another technique of propagation which works well with
some species is the use of single node cuttings, i.e., a
single bud left attached to its subjacent internode. Orton
(54) reports rapid propagation of red maple (Acer rubrum)
within 21-35 days with over 90 percent success using this
technique. We have obtained similar results using single
node cuttings of young coppiced sycamore (Platanus occidentalis)
under intermittent mist in a sand/peat rooting medium with
or without the basal application of rooting hormones such as
naphthalene acetic acid (NAA) or indole-3-butyric acid
(IBA), (9). In these experiments each node along the upper-
most lateral and terminal shoots of young 5-year-old sycamore
sprouts was planted in the sequence they occurred (apex to
base) to observe the rooting response and subsequent growth
of successive nodes along the shoot. Although over 90
percent of all the single node cuttings rooted, there was a
strong positive correlation in the number of roots and the
extent of root development, as well as subsequent shoot
growth, with increasing diameter of the basal internodes.
Differences in bud size, hormonal levels and initial supply
of reserve foods could all contribute to these observed
growth responses of excised nodes along the same shoot.
Peach clones (Prunus persica (L.) Batsch) were similarly
established by Robitaille and Yu (64) with 70 percent success
using sprouted nodal cuttings, i.e., buds with leaves about
 113

50 percent expanded, dipped in 100 to 500 ppm of IBA and

placed under intermittent mist. The cuttings survived field
planting in early summer, grew well, and overwintered without
injury.
These modified propagation techniques may well provide
alternatives which could be used to advantage in establishing
clonal banks of vigorously growing phenotypes for forest
production. Horticulturists have long used similar methods
to produce millions of propagules of woody ornamentals for
commercial production. It does not require extensive lath-
house or Quonset-type plastic greenhouse space with misting
facilities and partially controlled temperature conditions
to produce 100,000 plants, or a series of such facilities to
produce a million or more containerized seedlings ready for
outplanting in a single growing season. Once adequate clone
banks are established, the rooting of single node cuttings
in small tubes in periods as short as 4-10 weeks could
permit essentially continuous production of planting stock
for commercial production of certain species.

2.3. Economic considerations using conventional or modified

propagation techniques
After many years of vegetatively propagating scores of
woody dicots by rooting cuttings or grafting, especially
among numerous horticultural cultivars, it is surprising how
little precise information is available on production costs
of plantable propagules. Traditionally, standard texts on
plant propagation are devoted to discussions of techniques
and procedures successfully used in propagating different
species or cultivars without any consideration of production
costs or the economics of establishing clonal lines (29).
This is understandable, however, when one considers that the
initial cost of producing individual fruit or nut trees is a
relatively minor consideration in the establishment and
maintenance of a widely spaced production orchard where
routine weed and pest control, fertilization, pruning,
harvesting, and marketing costs, plus occasional complete
crop failures, determine differences between profit and loss
114

of annual fruit or nut crops. Even less information is

currently available on production costs of vegetatively re-
produced forest trees, but for different reasons. First,
because of the common difficulties encountered in propagating
most commercially important forest trees, essentially all
past research effort has been devoted to techniques to
overcome problems associated with the physiology of root
initiation, and secondly, with the exception of poplars and
willows, few attempts have been made to establish production
forests from clonal lines. Furthermore, the widely contrast-
ing differences in management objectives between horticultural
crops and the yields of wood or fiber, under long-term
rotations adds to the complexity of obtaining reliable
economic data for forest crops.
Some of the best estimates available on the costs of
producing plantable seedlings from rooted cuttings have been
made using coniferous species and in these instances the
cost is 2 to 3 times more expensive than seedlings produced
from seed. However, as pointed out by Kleinschmit et al.
(40) and Rauter (62) the initial highe~ costs of rooted
cuttings may be more than justified economically if the
growth superiority of cuttings is 10 percent or more than
that of seedlings where the non-additive genetic component
has been fixed through cloning. Furthermore, one should
consider that genetically improved species which can be mass
produced by rooting small nodal cuttings placed directly
into tubes or other types of plantable containers could sig-
nificantly lower the costs of production by using a one-step
procedure which permits mechanized planting of containerized
stock. More detailed estimates of the costs of producing
clonal material (plantlets) via tissue culture techniques is
given in Section 4.1.

3. VEGETATIVE PROPAGATION VIA TISSUE AND ORGAN CULTURE

3.1. Brief historical account of organogenesis in woody dicots
There is no need at this time to review all previous
research leading to organogenesis in woody angiosperms,
 115

i.e., bud and root differentiation leading to plantlet

formation in vitro. Several excellent reviews are already
available g iving detailed accounts of these developments
including the potentialities tissue culture techniques offer
in programs of tree improvement and production forestry (see
Chapters on vegetative propagation in this volume and other
reviews (7, 8, 21, 70, 88)).
The first reports of organogenesis in tree tissue
cultures were made by Gautheret (27) who obtained formation
of adventitious buds in cambial cultures of Ulmus campestris.
He demonstrated the requirement of an exogenous source of
sugar to obtain buds, that light improved vigor and growth
into leafy shoots, and that high concentrations of indolea-
cetic acid (IAA) inhibited bud formation. Later Jacquiot
(34, 35, 36, 37) made further observations on similar phenomena .
It should be noted that some of the trees used by Jacquiot
were up to 180 years of age. He established that cambial
cells from both trunk and roots could differentiate buds,
and showed that inositol was a limiting factor for bud
formation in summer, while in winter adenine appeared to be
one of the limiting factors. Roots were obtained from U.
campestris cultures with NAA added to the nutrient medium,
but buds were not formed in the presence of NAA . These
observations were interpreted to indicate that organogenesis
in vitro appeared to be influenced by at least two antagonistic
factors. These observations are of historical interest
inasmuch as they were made several years prior to the discovery
and identification of cytokinins, and, in fact, helped to
lay the foundation for many of the experimental systems used
today to obtain organogenesis in woody angiosperms.
Mathes (49) was probably the first researcher to achieve
growth of both shoots and roots in callus cultures of Populus
tremuloidesi however, no indication was given that the
shoots and roots were connected to form plantlets. A few
years later, Wolter (89) showed that shoots could be initiated
on callus of Populus tremuloides by adding 0.5 ~ g/ml of
benzyladenine (BA) to the basal medium in the absence of
116

auxin, and the shoots could be rooted on Wolter's basal

medium. Winton (85) cultured triploid Populus tremuloides,
and other members of Populus, and obtained plantlets.
Shoots were often produced on callus with 0.05 Wg/ml BA
using Wolter's basal medium. In contrast to other systems,
roots were obtained with 0.04 wg/ml 2-4-dichlorophenoxyacetic
acid (2,4-D) and kinetin at 1.0 Wg/ml. These earlier reports
(49, 89, 85) while of historical importance, left some doubt
as to the viability and degree of abnormality found in the
regenerated plants. Later, Winton (86, 87) demonstrated
that normal tree seedlings could be obtained from callus
cultures in vitro, although few in numbers.
Since these earlier studies were made on regeneration
in Populus, just slightly over a decade ago, much progress
has been made because of increased interest among workers in
this field and the real need and utility of reproducing
trees by vegetative means. Table 1 lists over 40 species or
cultivars of dicotyledonous trees in which bud or root
differentiation has been obtained leading to the formation
of plantlets or whole plants. From Table 1 three notable
features stand out: (1) almost all reports of plantlet
formation have occurred in the last decade and well over 50
percent of these within the past 2-3 years; (2) plantlet
formation has been highly variable with regard to numbers
produced, i.e., few studies have led to developing a workable
system for mass cloning of forest trees in contrast to
systems currently in use for certain vegetable or ornamental
plants; and (3) the majority of plantlets in forest trees
have been produced from young, juvenile material. Most
workers thus far have used either mature embryos or portions
of embryos excised from seed, or organs from young aseptically
germinated seedlings such as portions of cotyledons, leaves,
petioles, hypocotyls, axillary buds, or shoot apices. The
reason for working with juvenile material is obvious, because
it provides a system that is partly successful, while tissues
or organs from older trees are commonly refractive to
treatment because of "aging effects" mentioned earlier.
 117

Young, juvenile material is more responsive to cytokinins,

gibberellins, auxins, inhibitors, and other growth factors
than older tissues, probably because that portion of the
genome controlling organ formation in early stages of develop-
ment is less repressed or more capable of being derepressed
by hormonal treatment than in older more highly differentiated
cells. Even in cultures capable of undergoing organogenesis,
individual cells intermixed or lying adjacent to other cells
undergoing cell division and organized differentiation, are
incapable of taking part in organ formation. In the terminology
of today, some cells are "permissive" while others are "non-
permissive" with regard to becoming organogenic.
In spite of the difficulties with older perennial
plants progress is being made with these plants today because
more and more researchers are turning their attention to
them (see Chapter 13). Just a few years ago we were unable
to grow continuously the cells or tissues of trees on chemi-
cally defined media, now cells of many mature trees may be
easily grown as callus or suspension cultures for extended
periods on relatively simple defined media. In addition to
the possibility that such cells may, after several generations
on enriched media, become rejuvenated and undergo organogenesis,
there are other alternative approaches to overcoming problems
of aging. First, there are indications that cells and
tissues associated with sexual reproduction in older trees
such as nucellar tissue, ovules, or somatic anther cells,
may be induced to undergo organogenesis more easily than
other parts of the same plant. Likewise, the release of
basal root suckers or epicormic shoots along the bole which
appear to revert to a more juvenile condition as mentioned
earlier provide a good source of material for initiating
cultures. Furthermore, little research has been attempted
thus far using younger root tissues of older trees as explant
material for plantlet formation. Several forest trees
naturally produce root buds and lateral roots of certain
species have been used to propagate difficult to root species
(10) .
118

3.2. Types of cultures and their application to large scale

commercial propagation
Basically there are three major types of cultures one may
use leading to plantlet formation or regeneration of intact
plants, namely (1) callus cultures, (2) organ cultures, or (3)
cell suspension cultures leading to embryogenesis. All have
been used to varying degrees of success experimentally and some
appear to hold considerable potential for mass propagation on a
practical basis.
3.2.1. Callus cultures. Callus cultures may be started
from any type of explant material possessing parenchymatous
cells capable of renewed cell division to form an unorganized
mass of proliferating cells. From such cultures one attempts
to manipulate the culture environment (both nutritionally and
physically) to induce shoot or root differentiation hopefully
followed by some degree of extension growth. In general,
cytokinins as a class of growth promo tors appear to have some
role in stimulating bud or shoot formation and growth; whereas
auxins tend to favor root differentiation in cultures capable
of undergoing organogenesis. Cytokinins and auxins thus fre-
quently appear to be antagonistic to each other, at least at
physiological levels where either one may inhibit or reduce
the commonly ellicited morphogenetic response of the other .
Such interactions depend, in large part, upon the sensitivity
o f cells and tissues in culture, the levels of naturally occur-
ring inhibitors or growth promoters present, as well as many
other nutritional and biochemical factors (see Chapters 9, 10,
and 11).
From a practical standpoint one usually attempts to induce
bud differentiation and shoot growth in callus cultures followed
by transfer of excised shoots to one or more rooting media with
or without auxin to obtain root formation and thus an intact
plant. Under some conditions where only roots can be induced
to form from callus cells, one may transfer excised roots to a
bud induction medium. In actual practice this procedure is
seldom used, apparently because of the low incidence of bud
differentiation on roots, nevertheless for some species
 119

prone to form root buds under natural conditions the technique

may be worthy of consideration.
Although plantlets have been produced frequently from
callus cultures (Table 1) the practical use of this approach
is restricted to species or cultivars possessing capabilities
for high incidence bud differentiation and subsequent shoot
growth (Figures 1 and 2).

eM

FIGURES 1 and 2. Shoot formation from callus cultures. Fig. 1.

Multiple shoots from Liquidambar styraciflua L. from shoot tip
callus. Fig. 2. Differentiated shoots from shoot tip callus of
Robinia pseudoacacia L.
The use of callus culture for propagation has been in-
vestigated in detail by Berbee and coworkers (3, 4, 43).
Their primary objective was to attempt to circumvent poplar
decline in selected Aigeiros poplar clones by the combined
use of tissue culture and heat treatment. The general
approach was to obtain callus from shoot tips, regenerate
shoots and finally root the shoots. The plantlets were
grown to about 50 cm in size then established in mother
plant blocks in the nursery for observation. There were
several practical problems. No one series of media was
successful for the culture of all clones, and sometimes only
one third of the callus cultures produced shoots. However,
sufficient clones were established to permit comparisons
between the parent clone and the in vitro derived subclones.
There were significant differences in heights of some
120

subclones from the parent clone, as well as differences

between subclones derived from calluses from the same shoot
tip, or even those from the same callus. Chromosome counts
made on the shoot tips of some subclones were within the
normal range, however, 8 of 46 subclones ranged from haploid
to tetraploid, with some aneuploids. In another experiment
200 subclones were obtained from callus cultures from 7
different Aigeiros clones and all appeared to be virus
symptomless although none of the plantlets were virus indexed.
It is significant that sufficient numbers of plantlets could
be produced from callus culture to establish a field study
of this sort.
In other studies, Chalupa (12, 13) has produced numerous
Populus plantlets from callus. Up to 86% of the callus
cultures differentiated shoots. Rather than root the adven-
tive shoots on nutrient agar, he used sterile perlite and
sand or perlite, or sand and peat as a rooting substrate
which produced better root development. The plantlets grew
to young trees in pots. Later Chalupa (14) compared callus
derived plantlets with the parent ~rees. The growth rate of
plantlets from the same mother tree was similar, as were
color, form and size of leaves, and photosynthetic rates.
The chromosome number of the plantlets was the same as for
the mother tree. Thus, in this case the variability observed
by Berbee and his coworkers for callus regenerated plantlets
was not found.
Another example of callus culture to be considered is
the system of Riou et al. (63). Their approach was to
obtain friable callus of Populus euroamericana cv. "robusta" ,
and place it into liquid suspension culture. The free cells
of the suspension were obtained by filtration and centrifuga-
tion and plated onto agar in petri dishes. The colonies
that developed were transferred to tubes for shoot differen-
tiation, then the shoots excised and rooted. Each step
required a different medium. One can only wonder if each
colony represented a distinct single cell derived clone and
also if all the shoots from a callus were identical. It is
 121

unfortunate that no data were given on the variability

present because the use of callus from suspension cultures
could have much utility in developing a system for mass
producing regenerated shoots.
Plantlets have been obtained from callus cultures of
angiospermous trees other than Populus, e.g., Ulmus campestris
(13), Ulmus americana (22) and Acacia koa (67), but one of
the most interesting variations reported is the production
of birch plantlets by Huhtinen and Yahyaoglu (33) and Huhtinen
(32). For these experiments, they used an early-flowering
strain of Betula pendula. These flower within their first
year and bear only reproductive buds. A cambial callus was
initiated on MS medium plus 25 ~ g/ml IAA and 0.5 ~ g/ml

kinetin. Both roots and shoots were initiated independently

on this callus. Shoots were excised and rooted on Murashige
and Skoog's (MS) medium with 0.1 ~ g/ml 2,4-D. The plantlets
were potted, grew rapidly and initiated many lateral shoots.
In five months male flowers started to develop and the
pollen appeared normal; however, there were noticeable dif-
ferences from the parent plants. Most outstanding was the
observation that fast-growing plantlets produced some vegeta-
tive buds and could continue vegetative growth after flowering.
The parents had produced only reproductive buds. By virtue
of the early-flowering characteristic, which was maintained
by all the plantlets, it should be possible to do genetic
studies and to obtain a better understanding of genetic
changes that occur when a tree is propagated via callus
culture.
In view of considerable success in recent years in
producing plantlets from callus cultures of several trees,
especially among various species or cultivars of Populus, it
is somewhat surprising that a workable system for mass
propagation has not been forthcoming using this approach.
Some of the most obvious difficulties arising from the
use of callus cultures to regenerate forest tree for commer-
cial production lies in (1) the sporadic incidence of bud
and shoot formation observed in most species even on various
122

types of media, (2) length of time frequently required to

obtain buds or shoots from the initial explants (months in
many species studied), (3) variability in rooting response
of excised shoots, and (4) depending, in part upon the
length of time in culture prior to shoot differentiation,
increases in genetic aberration of one sort or another
leading to subsequent differences in growth response and/or
expression of desirable morphological traits.
3 . 2.2. Organ cultures. Several types of organ cultures
have been used to study organogenesis and plantlet formation
in dicotyledonous trees. In the broadest sense explants of
leaves, cotyledons, hypocotyls, or portions of embryos,
reproductive structures, shoot and shoot apices, as well as
terminal and axillary buds, would all fall into the category
of organ culture. Thus different types of organ explants
have been used by different workers to study specific problens
of organogenesis and regeneration (Table 1) (Figures 3 and
4)

Figures 3 and 4. Regenerated shoots from organ cultures.

Fig. 3. Multiple shoots from young leaf of Paulownia tomentosa
Steud. Fig. 4. Bud and shoot formation from excised root of
Robinia pseudoacacia.
Because of recent successes in mass propagating young,
and to some extent older, mature trees by shoot tip or
axillary bud cultures and the potentialities this approach
offers to commercial production of plantable tree seedlings,
 123

the remainder of this section will be devoted to a discussion

of the general procedures being used. The approach requires
surface sterilization of shoot tips or buds, followed by
leaf or bud scale removal, excision of the shoot apex with
attached young leaf primordia, and explanting it to an
appropriate medium to obtain continued development of
lateral meristems and the outgrowth of small extended shoots.
Young shoots with extended internodes are then subdivided
and subcultured to multiply the number of shoots required
for a given clone before being transferred to a rooting
medium for plantlet formation. This technique is essentially
an extension of conventional propagation techniques using
aseptic culture and is frequently referred to in the horticul-
tural literature as "micropropagation".
Whitehead and Giles (84) investigated the application
of rapid micropropagation with axillary buds of Populus
nigra 'Italica,' ~. deltoides x ~. nigra ('Flevo') and ~.

yunnanensis on a modified MS medium with 0.2 ~ g/ml BA.

Within 4 weeks bud break had occurred and initial shoots had
lengthened enough to be cut into 5 rom sections and placed
again on the same medium. Adventitious buds started to form
and the culture was transferred to a modified MS medium with
0. 1 ~ g/ml BA and 0.02 ~g/ml NAA. After 6 to 8 weeks 120-220
shoots had been formed for each bud explanted. These shoots
could either be rooted or used for the production of more
shoots. Three months after rooting, the trees were l-l~

meters tall. They estimated that one million plantlets per

bud per year could be produced using this approach.
Christie (18) likewise obtained plantlets from the pro-
liferation of buds from the shoots of Populus alba, Populus
tremula and Populus tremuloides. After the initial growth
of shoots from the buds, the shoots were subcultured to
increase the multiplication rate to 10 shoots/culture/month.
Following rooting in agar, the cultures were placed in a
greenhouse for 2 days to adapt them to its light and tempera-
ture regime. Potted plantlets were intermittently misted
for 7 to 14 days under long photoperiods and ninety percent
124

of the plantlets survived. Prior to field planting, the

plant lets were held in the greenhouse until they reached
about 10 cm in height and became adapted to outside conditions.
Height growth of 1.5 to 2 meters was obtained in 3 months
after ?lanting and all plants appeared identical to stock
plants.
Chalupa (15) has extended the regeneration of plantlets
to older trees from axillary buds of Ulmus campestris, ~.

scabra, ~. effusa, Quercus robur, Fagus silvatica, Populus

tremula, Populus euroamericana cv. robusta, Populus nigra
var. typica and Populus alba. The buds were obtained from
trees 10-20 years old. He found the shoots could be rooted
directly in a peat-perlite mix and transplanting the plantlets
to soil resulted in up to 80 percent survival.
Using a slightly different approach, Sommer and Brown
(70), explanted buds of young (lO-year-old) tulip poplar
(Liriodendron tulipifera) and sweetgum (Liquidambar styraciflua)
trees in mid-February, when winter dormancy had been completed.
They were surface-sterilized, the bud scales removed, and
the exposed shoot tip placed on modified Risser and White's
or modified MS media. Bud opening and growth occurred with
little delay. After transfer to fresh media for bud growth,
the buds were transferred to modified Morel's medium (76)
with lBA for root initiation. After two weeks the cultures
were transferred to Risser and White's medium without
auxin. A few buds from each species rooted readily, but no
attempt was made to mass propagate either species for commer-
cial use (Figure 5). Brown (9) also reported the formation of
numerous plantlets obtained from micropropagation of shoot tips
or axillary buds of several commercially important tree species
(see Table 1).
Gupta et al. (28) have reported the propagation of 100-
year-old teak trees by starting with the culturing of terminal
buds and axillary buds on an MS media with 0.1 ~ g/ml kinetin
and 0.1 ~ g/ml BA. After shoot initiation and elongation,
rooting was accomplished by transfer to White's medium with
lAA, lBA and indolepropionic acid (lPA) each at 2 ~ g/ml for
 125

48 hours then to White's basal medium. Rooted plant lets

were subcultured and additional shoots and plantlets obtained
from their axillary buds. The use of both kinetin and
benzyladenine appears to be particularly important in obtain-
ing the outgrowth of preformed buds of teak.

Figures 5 and 6. Shoot tlP cultures of young hardwood trees.

Fig. 5. Leaf and shoot formation from terminal shoot of
Liriodendron tulipifera L. Fig. 6. Single shoot from shoot tip
of Prunus serotina Ehrh.
In another study using Asiatic white birch (Betula
platyphylla cv. schezuanica) McCown and Amos (52) report
that shoot tip and nodal explants of this species placed on
Gresshof and Doy's nutrient agar supplemented with 4~M

BA produced numerous actively growing shoot cultures within

6 months. Shoots could then be easily increased in number
by subculturing monthly followed by subdivision of elongating
shoots. After subculturing, 20-30 utilizable shoots could
be harvested from each culture every 6-8 weeks for rooting.
These shoots rooted with 100 percent success within 2 weeks
(even without hormone treatment) in a 1:1 peat/perlite
mixture in a warm (30-35 0 C) high humidity (80%) environment.
After a period of acclimation in a greenhouse where they
were gradually exposed to full sunlight, the young plantlets
could be treated like tree seedlings in commercial production.
Although these experiments used explants taken from seedlings,
the authors claimed that some success was achieved with
126

shoot tips from mature trees. They indicated, however, that

the older mature buds would have to become "rejuvenated"
over time to a more juvenile physiological state. using the
shoot tip technique, these workers suggest that the rate of
multiplication is adequate for commercial production. \vith
an average production of 20 shoots per culture tube in a 6-
8 week period, one could generate 4000 propagules per square
foot of culture shelf space per year or one half million
propagules with only 125 square feet of culture shelf space.
This, of course, constitutes only the space required for
propagule production and not space for transplanting and
hardening off of seedlings in containers prior to field
planting. Even a more conservative estimate of obtaining 5
shoots per culture every 12 weeks with a 70 percent survival
rate of plantlets produced, one could still produce annually
a million progagules (ca. 20,000 plants per week) with less
than 1200 square feet of culture shelf space (Table 2).
Some of the operational difficulties associated with this
phase of vegetative propagation are discussed in Section 5.
The economic feasibility of this system will be considered
after a brief account of another alternative production
system is given, i.e., embryogenesis, which offers even
greater potentialities for production forestry in the future
and one with which some initial success has been attained.
3.2.3. Plantlet formation via embryogenesis in cell
suspensions. Throughout the literature there are many
claims of embryogenesis in tissue culture, some of which are
of doubtful authenticity. Table 3 presents several accounts
of embryoid formation in woody dicots, none of which has
presently led to commercial utility with the possible excep-
tion of the system of high frequency somatic embryogenesis
developed by Sondahl and Sharp (72) in cultures of Coffea
arabica.
Briefly, the earliest report found on embryogenesis in
culture of a forest tree is in Santalum album L. (59). The
embryo ids were easily separable from the parent plant and
 127

readily put out a root, but developed very poorly fasciated

embryonic shoots.
Hu and Sussex (31) reported on the development of
embryoids on the cotyledon of Ilex aquifolium. The cultures
were unusual in that the embryoids developed on abnormal
cotyledons of zygotic embryos that failed to develop shoots
on a basal Linsmaier and Skoog medium. Callus formation was
not apparent and anatomical studies showed no vascular
connections to the parent tissue. Anatomically the embryoids
appeared normal and under specific conditions could be
germinated.
Adventive embryogenesis in Theobroma cacoa has been
studied by Pence et al. (55, 56). Somatic embryos were
obtained only from immature zygotic embryos, but a higher
percentage of embryogenic cultures were obtained in liquid
medium than on nutrient agar. Embryoids were found to
differentiate in two different ways. In one case ovoid
outgrowths were formed on the edges of the cotyledons and
multicellular hair-like structures were differentiated on
their surface. The terminal cell(s) then commence division
leaving a few stalk cells. Developmept of the embryoid was
followed histologically to the point where cotyledons and
procambium had formed and no vascular connection to the
mother tissue was evident. Less frequently, ernbryoids
differentiated from internal meristematic tissue of the
cotyledons similar to the ovoid outgrowths except develop-
ment was direct to fully formed embryoids.
Sondahl and Sharp (72) have developed a system for high
frequency somatic embryogenesis (HFSE) in cultures of Coffea
arabica. The embryoids obtained developed into normal
coffee plantlets. Yields are in the range of 50-100 embryoids
per 30 ml culture bottle. Up to 60% of the cultures show
HFSE. The system for obtaining HFSE and low frequency
somatic embryogenesis (LFSE) both start with leaf tissue on
a "conditioning medium" followed by growth on an "induction
medium". For HFSE 2,4-D cannot be replaced in the "condition-
ing medium" by other auxins without greatly reduced success.
128

S?ace does not allow a complete consideration of the systems;

however, we might note the MS medium inorganics and organics
are greatly modified. Also in the "induction medium" kinetin
to auxins (usually NAA) ratio is high, but their absolute
concentrations are lower. Detailed histological and morphologi-
cal studies of HFSE and LFSE tissues were conducted (73, 74).
Other interesting studies on non-zygotic embryogenesis
in woody plants are those of Radojevic, (57) (58). Corylus
avellana and Paulownia tomentosa embryogenic callus was
obtained from zygotic embryos, and from unfertilized ovules
in the case of P. tomentosa. In the presence of 2,4-D
plantlet formation was inhibited, but more so for C. avellana
than P. tomentosa.
More recently, Sommer and Brown (71) obtained adventive
embryos from callus of young sweetgum seedlings on nutrient
agar using Blades basal medium, followed by the formation of
embryoids from cells in suspension culture (Figures 7 and
8). There was considerable variation in the embryogenic
response and much research remains to be done before the
system can be developed for practical use .

eM

Figures 7 and 8. Embryogenesis and plantlet formation in

Liquidambar styraciflua L. Fig. 7. Young embryoids differen-
tiated in suspension culture from hypocotyl callus then trans-
ferred to Blades nutrient agar. Fig. 8 Later stage of plantlet
development from embryoid.
 129

4. ECONOMIC CONSIDERATIONS
4.1. Cost comparisons of seedlings produced by tissue culture
techniques versus seedlings produced from seed
In attempting to make direct economic comparisons
between different systems of producing plantable seedlings
for reforestation one is always confronted with making
certain assumptions within the limits of recognized constraints.
In order to obtain some reasonably reliable comparisons
between seedlings produced via tissue culture and those
produced by conventional nursery techniques one is confronted,
with three broad considerations, namely: (1) capital
outlay in the form of land, buildings, facilities, and
equipment; (2) total direct production costs including
supervisory personnel, labor, taxes, insurance, supplies and
expendibles, equipment operation, heating and cooling,
lighting, and etc. which continue to escalate continuously;
and (3) the potential value of the product produced within a
given time period, in our case, the yield of forest products
at the end of the rotation.
For simplification purposes only, assume that capital
outlays and depreciation rates are comparable for both
systems even though the large acreages required for commer-
cial seed production in seed orchards, plus considerable
nursery acreage required for seedling production, offices,
warehouses for seedling packaging and storage, outlays for
heavy equipment, tractors and etc., would in all probability,
far exceed a tissue culture facility consisting of one
building containing approximately 3000 square feet of usable
space with a controlled temperature and light facility of
only 400 square feet, a small greenhouse (ca. 1200 sq. ft.)
and attendant lathhouse space of ca. 20,000 square feet for
use in acclimating seedlings prior to planting. Furthermore,
to be more conservative we may also disregard the value of
the final product produced although by fixing the genotype
of the best selected genetically superior clones one could
certainly expect greater yields from cloned material then
130

from first or second generation heterozygous seedlings from

clonal orchards (see Chapter 3).
Because all of the above mentioned economic inputs vary
so much with c~untry, location, management objectives,
species, yields, and final product produced, it appears
appropriate to focus more attention to comparisons of direct
production cost between plantable seedlings produced as (1)
bare root, 1-0 nursery stock, (2) containerized seedlings
produced from seed, and (3) containerized seedlings from
shoot-tip culture or "micropropation" procedures. Production
costs for the first two systems can be readily obtained from
privately owned industrial nurseries and from published
reports on containerized seedling production. To obtain
some estimate of the latter we have analyzed the cost of
explanting, subculturing, and rooting shoots of tree species
in our laboratory at current (1981) prices. These data are
given in Table 4, and a relative comparison of production
costs for the three systems above is given in Table 5. In
addition, Table 6 gives the estimated cost of plantlet
production for several vegetable or ornamental plants which
include amortization of capital outlay for buildings and
equipment. In all cases, well over 50 percent of the total
costs of producing tissue culture plants involves labor used
in explanting and subculturing. Hence, a system of embryo-
genesis might easily reduce the total cost of labor by as
much as 60 to 80 percent of the amount currently used in
shoot tip culture.

5. PROBLEMS ENCOUNTERED IN PROPAGATION OF TREES USING

TISSUE CULTURE TECHNIQUES
The first prerequisite for the successful application
of tissue culture to vegetative propagation is a high fre-
quency system of organogenesis or embryogenesis leading to
rapid plantlet formation. Second, the plantlets produced in
vitro must survive transplanting, hardening off and perform
in the field as expected or better. Third, the use of
tissue culture should be compatible with or offer advantages
over conventional systems of propagation. Finally, the
 131

desirable characteristics or traits sought and fixed by

propagation must justify, economically or otherwise, the use
of tissue culture rather than conventional means of propaga-
tion. Several problems one most frequently encounters in
the practical application of tissue culture techniques are
briefly discussed below.

5.1. Inherent difficulties with trees

Most researchers involved in vegetative reproduction
using tissue culture techniques quickly become aware that
two of the most difficult problems encountered are generally
Common to most trees, namely: (1) inter- and intraspecific
genotypic variation in regeneration responses, and (2)
processes of aging. Both of these topics have been addressed
in Section 2.1 and 3.1 in this chapter and dealt with in
considerable detail in Chapters 3, 12, and 13. For emphasis
only, it is well to point out again that each species to be
cloned frequently requires the development of a system for
that particular species, i.e, what works well for Alnus
rubra does not, work at all for Alnus glutinosa. Unfortunately,
our experience over the years with closely related species
of conifers, and our recent research with several woody
dicots has been similar. In addition to interspecific
variation in cloning responses, one frequently encounters
widespread genotypic variation even within half-sib or full-
sib families. This latter source of variation still presents
considerable difficulty in developing practical systems of
mass propagation of selected phenotypes from a given seed
source. Fortunately, some progress is being made as more
and more workers turn their attention to obtaining high
frequency organogenesis or embryogenesis.
Although there are still many problems relating to
aging in propagating older trees there are many indications
that some of the difficulties can be overcome by using one
or more of the techniques mentioned in Sections 2.2 and
3.2.2 of this chapter and also in Chapters 6 and 13. Most
commonly these include (1) severe pruning or hedging; (2)
132

grafting onto juvenile root stocks prior to culture; (3) use

of stump sprouts or root suckers; and (4) use of older
shoot-tip cultures to initiate successive cycles of enhanced
shoot production.

5.2. Problems associated with transplanting and hardening off

of plantlets
Plantlets produced in vitro must be acclimated or
hardened off to withstand the less humid, warmer temperatures,
and higher light intensities existing outside the high
humidity environment where they were formed. Plantlets
removed from culture are unusually susceptible to desiccation
and wilting because of their highly succulent nature and
excessive water loss before roots can become physiologically
functional after transplanting. Nevertheless, there is
extreme variation in species response to transplanting and
adaptation to stressful environments both in (1) physical
parameters of the new environment (humidity, temperature,
and light) as well as (2) the time period required for the
young plants to develop adaptive anatomical and morphological
features following transplanting. For example, Berbee et
al. (4), Chalupa (15) and McCown and Amos (52) were able to
obtain rapid root formation and excellent survival of poplar,
birch and other hardwood shoots excised and transplanted
directly from agar cultures into peat/perlite or other
potting mixtures under warm and highly humid conditions
(humidity 80 % or higher); conversely, Skolmen and Mapes (68)
experienced failures in many initial attempts to obtain
autotrophic development of young Acacia plant lets (possessing
roots formed on nutrient agar) when transferred to a perlite
or peatmoss-vermiculite medium maintained under intermittent
mist or enclosed in polyethylene bags. Plants removed from
aseptic media and transferred to the rooting medium under
polyethylene rapidly desiccated and died of moisture stress
even though microscopic observations showed good vascular
connections between shoots and roots. Similarly all trans-
planted plantlets maintained under intermittent mist succumbed
 133

upon removal of the mist. To induce functional roots,

plantlets were removed from agar and grown in a hydroponic
medium (Hoagland's solution) for a month before being trans-
ferred to a potting medium resulting in 83 percent survival.
Hence, these workers found it necessary to develop a 4 step
procedure for successful acclimation of Acacia plantlets
prior to field planting. These were: (1) transfer of
plantlets from agar to Hoagland's solution in flasks with
tops covered by polyethylene for one month, (2) transplanting
to a mi x tur e of peatmoss, perlite and vermiculite, fertilized
monthly with liquid fertilizer, and covered with polyethylene
for 4-6 weeks after transplanting in the laboratory, (3)
transfer to a greenhouse after 2-3 months for further growth
under approximately 70 percent full sunlight and higher
temperatures (survival at this stage was about 77 percent),
and (4) after 1-2 months of rapid growth in the greenhouse
the plants were moved to an outdoor nursery for an additional
2 months prior to field planting.
Recently a process for transplanting and acclimating
sweetgum (Liquidambar) plantlets obtained from hypocotyl
segments of young plants as developed by Sommer (69) requires
essentially two steps. First, the young 4-S month old
plantlets with several well-formed roots are removed from
nutrient agar by careful washing and transferred to a peat-
perlite potting mixture in small containers covered with
either polyethylene or glass vials to maintain high humidity.
The bags or covers are gradually removed over a period of 1-
2 weeks during which time the initial roots have become
functional in water and nutrient uptake. The young plants
are maintained for 6-8 weeks under laboratory growth conditions
of approximately SOO f.c. of mixed fluorescent / incadenscent
light, at 2S~30C, and a lS-hour photoperiod. After the
shoots have reached 24-36 cm in height, 8 to 12 weeks later,
the plants are transferred to a greenhouse under full sunlight
with daily temperatures ranging from 20-3S o C. After 2-3
months and periodic fertilization they are transferred to a
lathhouse for continued summer growth and allowed to go
134

dormant prior to late fall or winter field planting. Survival

rates of up to 75 percent have been obtained.
From these and other observations it is apparent that
the process of transplanting and acclimation of hardwood
plantlets into plantable seedlings may vary from one extreme
to the other depending upon the system used and species re-
sponse to postculture manipulation. Different species re-
generated by tissue culture techniques will require specific
methods of handling best suited to that species under a
given set of conditions.
Fortunately much information is now becoming available
on various methods of seedling containerization in reforesta-
tion and many of these techniques can be readily adapted to
establishment of tissue culture plantlets. Because hardening
off processes will likely be essential for most species, at
least for short periods of time, specially constructed high
humidity chambers possessing precise humidity and temperature
controls along with proper light intensity will be necessary
for maximizing seedling production in a tissue culture
facility. This may best be accomplished by a series of
specially constructed growth chambers programmed for periodic
movement of young plants to successively more stressed
environments until they can withstand lathhouse and/or field
conditions without injury. The space required for this
purpose need not be a costly constraint in the process of
production considering that 20,000 plantlets per week can be
moved through a facility requiring a shelf space of less
than 1000 sq. ft. (see Section 4.1). The capacity, of
course, depends upon production objectives and if modular
blocks of controlled environment space can be placed on line
as needed. Energy required for cooling and lighting could
be minimized by construction of appropriately insulated or
partial underground units provided with full overhead sunlight
in the final acclimation stage prior to transfer to outside
conditions.
 135

5.3. Production costs

The cost of mass producing seedlings for production
forestry by tissue culture techniques is of much concern to
researchers and foresters alike. As indicated in Section
4.1 mass generation of plantlets, transplanting, and hardening
off operations prior to establishment in the field are
highly labor intensive, and require constant superivison
from start to finish. At least 50 to 80 percent of all
direct production costs can be attributed to technical and
skilled labor involved in plant collections, sterilization
procedures, explanting, transplanting, and maintenance of
cultures. Added to this are managerial and administrative
costs requiring knowledgeable persons in the area of tissue
culture technology and personnel management.
Although current costs of producing plantable seedlings
are at least 2 to 3 fold greater than present nursery produc-
tion costs, the economics of seedling production depend on
the final value of the product produced in both quality and
quantity. Even though little specific data are available on
the value of genetically improved seed some industrial
companies value improved seed from seed orchards (pine) at
approximately $1000.00 per pound (47). If a pound of seed
resulted in 20,000 plantable seedlings for the species in
question, then the seed costs alone would amount to $50.00
per thousand plants. Add to this another $50.00, or more,
for direct production costs of each thousand plants, then
the current estimated price of $123.00 per thousand (see
Section 4.1, Table IV) for tissue culture plants becomes
less formidable than it first appears. Certainly, some of
the current concern over production costs will be alleviated
just as soon as systems for embryogenesis are developed for
genetically improved strains.

6. FUTURE OUTLOOK
6.1. Use of shoot-tip cultures
Of the present strategies used for vegetative propagation
of economically important dicot trees, the use of shoot tip
136

cultures appears to hold most promise for immediate applica-

tion. The method stands out among others because of (1) the
high frequency of organogenesis, (2) an acceptable level of
genetic stability, (3) its applicability to a large and di-
verse number of species, (4) some success in propagation o f
older trees, and (5) its economic feasibility.
It now appears possible to extend this method of micro-
propagation to commercial regeneration of high value hardwoods
selected for specific genetic traits such as wood quality,
form, and growth rate if proper attention and expertise is
given to developing a system for individual species. The
method should also hold considerable promise in cloning
genetically improved strains of young, rapidly growing
hardwoods to enhance biomass production for fiber, che mical
feedstocks and fu e l under short rotation systems of forest
management.

6.2. Potentialities of Embryog enesis

The production of embryoids in tissue culture potentially
has sev eral advantages over organogenesis as a practical
means of propagation. For example, a method of producing
embryoids in suspension or liquid shake cultures which
develop directly into normal intact plants as achieved in
the classical experiments of Steward et al. (77) with carrot
(Daucus carota) could immediately mak e mass cloning o f tree
species operational and economically f e asible. First, it
would bypass the necessity of timely and costly dissecting
and explanting individual shoot tips or other material to
obtain organo g enesis; secondly, it would alle v iate the need
to subculture numerous shoots to incre ase clonal stock; and
lastly, it would alleviate the f inal phase of subculturing
to produc e rooted plantlets. Thousands o f young e mbryoids
could b e mass trans fe rred to semi-solid media to de v elop
into s mall plants, or perhaps directly transferred to small
pre loaded tubling s, hardene d o f f, then later outplanted by
hand or machine into plantations. It has even been proposed
that e mbryoids could possibly be encapsulated and then
 137

handled as seeds. For these, and other reasons, researchers

in various laboratories around the world are presently
attempting to develop systems of embryogenesis for difficult
to propagate tree species.
One should not lose sight of the fact that less than
five years ago no one had produced sufficient plantlets from
tree species to be tested in field plantations. Less than
two years ago no one had produced embryoids from an economi-
cally important forest tree species which grew into normal
plants. Today we are talking about the economic feasibility
of producing annually a million or more plantlets of certain
species using 250 to 1000 square feet of laboratory space
prior to hardening off the young containerized plantlets in
an adjacent facility for field planting. One can only be
enthusiastic about continued progress in developing practical
systems for mass propagation of forest trees.

7. REFERENCES
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138

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 139

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140

46. LU CH, H CHANG, Y LIU 1978 Induction and cultivation

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52. McCOWN B, RAMOS 1979 Initial trials with commercial
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53. OKA S, K OHYAMA 1974 Studies on the in vitro culture
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54. ORTON ER 1978 Single-node cuttings: A simple method
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55. PEN~VC, PM HASEGAWA, J JANICK 1979 Asexual
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Plant 13: 200-206 -----
62. RAUTER RM 1974 A short-term tree improvement programme
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 141

66. SITA GL, NV RAGHAVA RAN, CS VAIDYANATHAN 1979

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142

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Table 1. Some Dicotyledonous Tree Species from which Plantlets have been Regenerated from
Tissue or Organ Culture

Tree Year
Species age a Explant Source Cultured Reference

Acacia koa Gray S Shoot tip 1976 (67)

Acer rubrUm L. S Shoot tip 1980 (9)
axillary buds
Aesculus hippocastanum L. R Anther (haploid) 1978 (57)
Alnus glutinosa Gaertn. S Shoot tips 1980 ( 9)
Alnus rubra Bong. S Shoot tip, 1980 (9)
buds hypocotyls
Betula pendula Roth S Sterns 1974 (33)
Betula pendula Roth R Anthers (haploid) 1978 (32)
Betula platyphylla cv. S Shoot tips 1980 (52)
schezuanica
Broussonetia kazinoki Sieb. S Hypocotyls 1974 (53 )
Carya illinoensis S Axillary buds 1981 (41 )
(Wang) Koch
Castanea sativa Mill. E Embryonic axis 1980 (81)
Catalpa bignonioides Walt. E Shoot tips 1980 ( 9)
Corylus avellana L. E Adventitious buds 1978 (38)
Eucalyptus alba Reinw. S Hypocotyl 1975 (39 )
Eucalyptus grandis L. S Axillary buds 1975 (19)
Eucalyptus robusta Smith S Shoot tips 1980 (9 )
Fagus silvatica L. Y Axillary buds 1979 (15)
GIedItsia triocanthos S Shoot tip 1980 (9 )
var. inermis Willd.
Hevea brasiliensis R Anther (haploid) 1978 (17)
--guell.-Arg.
Liquidambar styraciflua L. S Shoot tips 1980 ( 9)
Hypocotyls
Liriodendron tulipifera L. Y Terminal buds 1980 (9 )
Paulownia taiwaniana R Ovules 1978 (25 )
Hu and Chang ~
w
Table 1. Cont'd t

Tree Year
Species age a Explant Source Cultured Reference

Paulownia tomentosa Steud. S Shoot tip, axillary 1980 (9)

buds, petiole
Populus alba L. Y Axillary buds 1979 (15)
Populus alba L. var. typica Y Axillary buds 1979 (15)
Populus x canadensis Moench. Y Stern-tip 1972 (3)
Populus canescens Smith S Cambium, shoot tip 1974 (12)
Populus euroamericana S Cambium, shoot tip 1974 (12)
(Dode) Guinier
Populus euroamericana Y Axillary buds 1974 (12)
cv. 'robusta'
Populus nigra L. R Anther (haploid) 1975 (83)
Populus nigra cv. Y Twigs 1973 (79)
'Italica' DuRoi
Populus tremula L. S Sterns 1971 (87)
Populus tremuloides l1ichx. S Sterns 1970 (86)
Populus tremula L. Y Axillary buds 1979 (15)
Populus (hybrids) R Anther (haploid) 1978 (46)
Populus (species and hybrids) R Anther (haploid) 1980 (91)
Quercus robur L. Y Axillary buds 1979 (15)
Robinia pseudoacacia L. S Shoot tips, axillary 1980 (9)
buds
Salix babylonica L. Y Axillary buds 1977 (44)
santalum album L. E Embryos 1971 (61)
Santalum album L. S Hypocotyls 1978 (60)
Santalum album L. Y Shoot tips, stern 1979 (66)
Tectona grandTs L. S,H Shoot tips, stern 1980 (28)
cotyledons, terminal
and axillary buds
Ulmus americana L. S Hypocotyls 1975 (22)
Ulmus campestris L. Y Twigs 1975 (13)
Ulmus effusa Wild . Y Axillary buds 1979 (15)
Ulmus scabra Mill . Y Axillary buds 1979 (15)

a E - embryo; M - mature; R - reproductive organ; S - seedlings; Y - young tree.

 145

Table 2. Stages of Plantlet Production and Number of Explants

to be Cultured or Transplanted to Produce 20,000
Plants/Week (ca. 1 million plants/year)a

Number Weeks Shoots Fold

Stage Type Explant Explanted Duration Produced Increase

1 Shoot tips 100 12 500 5

2 Shoots 1,000 12 5,000 5
(divided once)
3 10,000 8 20,000 2
4 Rooted shoots 20,000 4 1

a For species of Populus, Acer, Robinia or other woody dicots

induced to proliferate numerous shoots from shoot tip cultures
followed by high rooting incidence (70-80 percent) under
laboratory conditions. In actual practice one would need to
produce approximately 25,000 shoots at stage 3 in order to
obtain 20,000 containerized plantlets.
 .I:>
0\

Table 3. Some Dicotyledonous Species from which Embryoids have been Regenerated from Tissue
or Organ Culture a

Species Explant Source Culture Type Media b Reference

Coffea arabica L. leaf callus MS (72)

Coffea canephora L. young stem callus HE (75)
Pierre ex Froehner
Corylus avellana L. immature callus MS (58)
embryos
Ilex aquifolium L. immature embryo LS (31)
embryo
Liquidambar styraciflua L . seedling callus BL (71)
Santalum album L. embryo callus WH (59)
Santalum album L. 20-year stem callus WH (66 )
MS
Paulownia tomentosa Steud. ovules callus MS (58)
Theobroma cocao L. immature embryo MS (55)
embryos

a This list is incomplete with respect to other cultivated woody plants, notably Citrus
Carica, Hevea, Vitis etc.
bMS - Murashige and Skoog; HE - Heller; WH - White; LS - Linsmaier and Skoog; BL - Blades
 147

Table 4. Estimated Direct Production Costs per Thousand Con-

tainerized Seedlings usin~ Shoot Tip Culture Tech-
niques (1981 U S Dollars)

Wages and Salary Costs Costs/1000

Explanting Labor $ 84.00
Supervision/administration S 20.00
Media Cost and Preparation $ 6.30
Glassware and Expendibles $ 2.60
Containers and Nutrients $ 5.00
Overhead Costs
Heating, cooling, lights, water, etc. $ 5.00
Total Direct Cost $122.90

a Does not include amortization of facilities and equipment.

These estimates are based on information and records main-
tained in a research rather than an operational commercial
facility. Data is based on a 5 fold increase in shoot
propagules during a 10-12 week culture cycle using easy to
root species such as Acer rubrum or Robinia pseudoacacia
(10) .
148

Table 5. Comparison of Direct Production Costs per Thousand

Plantable Sweetgum Seedlings using Three Different
Systems of Production (1980 U S Dollars)

Approximate
System Costs at Production Site
Bare root, 1-0
Nursery Stock From
Unimproved Seed $65.00 a
Containerized Seedlings
Using Unimproved Seed $75.00 b
Containerized Seedlings
From Shoot Tip Cultures $123.00 c

apersonal communication from an industrial pulp and paper

company in Georgia. Prices of Sweetgum seedlings produced
by State Forestry Organizations in the SE, USA are commonly
sold to landowners below actual costs and ranged from
$28.50 to $50.00/thousand. (Forest Resources Mgt. Tech
Note #4E. Cooperative Ext. Service, Mississippi State
University. 1980.
bFrom estimates of Vyse and Ketcheson (82) and Mann (47)
including 12 % annual adjustment for inflation since publi-
cation of papers in 1974 and 1977, respectively.
CEstimated costs by Brown and Sommer for Shoot tip cultures
of several hardwood species in a research laboratory not
designed for commercial production (10).
Table 6. Estimated Total Costs per Thousand of Tissue Culture Produced Vegetable
or Ornamental Plantlets Including Amortization of Facilities and
Equipment (1978 U S Dollars)

Plant Species Type Explant Type Plantlet CostS/Thousand

Lily (Lilium) bulb scales rooted bulbs $41. OOa
Broccoli flower buds rooted shoots $154.00 b
(Bras sica)
Boston Fern $10.00-140.00 c
(Nephrolepis)
Fig (Ficus) $40.00-220.00
Dracaena $50.00-140.00
African Violet (Viola) $100.00-140.00

aAnderson and Meagher (2)

bAnderson and Meagher (1)
CRange of prices given at Oregon State University Ornamental Short Course (1978).
Mimeographed handout.

~
\0
150

6. VEGETATIVE PROPAGATION OF EUCALYPTUS

R. DURAND-CRESSWELL, M. BOULAY, and A. FRANCLET

1. THE GENUS EUCALYPTUS

At the present time, Eucalyptus is of increasing importance for timber and
pulp and paper production throughout the world, and in plantations rates as
one of the most productive forest crops. Species of Eucalyptus have been
widely planted in South America, Africa, Asia, Spain, Portugal, Middle Eastern
countries and North America (Fig. 1).

Fig. 1. Adult Eucalpytus in the field; E. grandis.

 151

The genus Eucalyptus, of the Myrtaceae family consists of 445 species, 24

sub-species and 24 varieties (23) but 870 names are currently used, 115 of
which designate natural hybrids (47). Eucalyptus is endemic to Australia and
the islands to the north including Timor, New Guinea and the Phillipines (25).
It grows in diverse climates and soil types and can be found up to an altitude
of 1,850 metres in N.S.W. Australia. In fact, Eucalyptus grows in a variety of
soil types from sandy (with a saline content of 2-3%), to swampy or waterlogged
soils through rocky to dry soils (103). This ability of certain species of
Eucalyptus to grow in marginal soils is an advantage which, with its rapid
growth rate, must not be overlooked when considering the economic value of this
forest tree. Besides its commercial use for timber, paper pulp and essential
oils, Eucalyptus is employed as wind breaks and firebreaks and also for quick
coverage of the soil after forest fires and to prevent erosion. Some species
are becomming increasingly popular as ornamental plants.
However, the lack of cold resistance of Eucalyptus has limited its use in
plantations in England (52) and in France (46). Damage from cold is not a
simple phenomenon of a species being unable to tolerate a certain low tempera-
ture and then dying. The extent and severity of damage to the tree is affected
by four factors: absolute minimum temperatures, sharp falls in temperature,
frozen soil and cold blast. In France, AFOCEL (Association Foret Cellulose) has
started an intensive program to develop species suited to this country. They
use various plus trees of Eucalyptus, which were planted in France 20 to 30
years ago and have proven resistant to the climatic conditions. AFOCEL is
attempting the production of hybrids with both the qualities of cold resistance
and good growth potential. Selected trees of the cold-resistant species E.
gunnii and ~ pauciflora (female) are crossed with the vigorous species E.
dalrympleana and ~ delegatensis (male) (46). Since natural hybridization of
Eucalyptus has often been reported (106), artificial hybridization is a means
of producing a limited number of seeds. The selected hybrids must then be
multiplied by vegetative propagation.
Natural regeneration of Eucalyptus is mainly by seed and the breeding of
Eucalyptus is generally a slow process because of the length of their juvenile
phase before flowering. One characteristic of the genus Eucalyptus is the mor-
phological variation associated with ontogenetic development. Leaves of nearly
all species are heteroblastic with three distinct stages of development: juve-
nile, intermediate and adult (11). The time for change from juvenile to inter-
mediate to adult foliage varies with species (18).
152

2. MEANS OF VEGETATIVE PROPAGATION

All commonly recognized methods of vegetative propagation have been tried
with Eucalyptus and most have resulted in failure especially when applied to
adult tissues. This means that by the time a tree has reached the size and age
that it can be evaluated by foresters and geneticists, it has already passed thE
stage at which it can be propagated vegetatively.
Another problem encountered with the vegetative propagation of Eucalyptus is
plagiotropic growth. For example, in ~ cladocalyx F.v.M., if lateral branches
are used as grafts, the growth is always horizontal (83). Similarly, Cauvin
(pers. comm.) experienced plagiotropic growth with air layers of several Euca-
lyptus species.
The three main methods of vegetative propagation experimentally used with
Eucalyptus are: air-layering, grafting and cuttings.
2.1. Air layering
Naturally occuring aerial roots have been observed on adult trees of E.
robusta (64, 108), ~ camaldulensis (64, 74) ~ deglupta (137) and several
species of redgums (Ill). No correlation was found (108) between tree size and
root formation, although the lack of aerial roots in Australian stands was
attributed to climatic conditions.
Natural layering has also been observed in the field with trees that have
fallen over and have become partially buried (74).
In the practice of air-layering or marcotting, roots form on aerial parts of
plants after girdling when the point of injury is enclosed in a moist rooting
medium (70). This technique, which is often used with slow rooting species,
enables the shoot to be supplied with nutrients from the parent plant during
root formation. Twenty six Eucalyptus species have been reported (7) to have
been propagated by layering.
As with cuttings, the rooting of air-layers is affected by season (65) and
is most successful when using seedling tissues (~ fastigata, ~ botyroides, ~

bicostata, ~ saligna) (106) or epicormic shoots (~camaldulensis (75) and E.

grandis (71. Adult trees can be layered by grafting adult scions onto seed-
ling rootstocks and applying layers to the scion (71).
2.2. Grafting
Grafting has been the preferred method of vegetative propagation of forest
species and difficult-to-root horticultural species since, although more expen-
sive, it is generally more reliable than cuttings. Grafting in Eucalyptus is
used to preserve the flower buds on the scions so that experimentally-controlled
 153

cross-pollinations can be done to establish seed orchards and also to multiply

selected plants by grafting on a seedling rootstock so that a desirable geno-
type may be cloned. Grafting of adult tissues onto seedling rootstocks is also
being done (20, 61, 63) to obtain rejuvenation of the adult tissues and thus to
obtain rootable cuttings (Fig. 2).

Fig. 2. Rejuvenation by grafting: E. camaldulensis Denh. Franclet's experiments

1969.

Grafting techniques that have been successful with Eucalyptus are: approach
grafting, side grafting, splice grafting, cleft grafting, whip grafting, bark
grafting, ring-veneer grafting, herbaceous grafting, crown grafting and budding
(71).
Species of some systematic groups graft easily onto their own rootstock
whereas other species take better when grafted onto a rootstock of a different
species (108). In addition, the establishment of a graft union is not necessar-
ily related to the future growth and compatability of the graft. Delayed incom-
patability is common in grafts of Eucalyptus and makes this means of vegetative
propagation uneconomical. For example, in grafts of E. deglupta, there were no
incompatability symptoms until shortly before the death of the tree which oc-
cured in trees several years old (38). In addition, grafts of ~ grandis scions
onto ~ deglupta rootstocks died even though an effective graft union was es-
tablished and the scion had been growing actively for several months (71). Most
154

studies on grafting describe the incompatability symptoms but little is known

about the physiology of incompatability.
It has been suggested that with E. grandis (98) and with ~ deglupta (36)
rooting inhibitors found in the adult leaves and bark, m~y be involved in graft
incompatability. It has also been observed (7) that after exposure to air and
light, the cut surfaces of E. ficifolia form a substance that is incompatible
with the formation of a good graft union.
Several other factors reported to influence successful grafts are the number
of leaves on the rootstock (19), girdling of the scion wood before grafting
(112), vigour of the rootstock (112), tissue damage and the effect of air on
cut surfaces (7), and season (36, 56, 88).
2.3. Stem cuttings
The advantages of stem cuttings as a means of vegetative propagation are
that a large number of cuttings can be obtained from a single tree, the problem
of graft incompatability is avoided and rooting of cuttings is usually cheaper
than other methods of propagation such as layering and grafting (71). The suc-
cess in rooting Eucalyptus cuttings has been shown (4, 59, 88) to depend on the
following factors: a) heredity; b) age both of mother plant and the tissues in
the cuttings; c) position of the cutting on the mother plant; d) size of the
cuttings; e) trophic factors; f) season; g) microclimate for rooting; h) root-
ing medium.
In Eucalyptus, the general findings are that they are easy to propagate by
cuttings provided leafy cuttings are taken from very young seedlings or epicor-
mic shoots from the base of the tree (4, 37, 57, 58, 59, 65, 68, 84, 85, 88,
106, 107). For example, with ~ camaldulensis and E. transcontinetalis,
cuttings rooted if taken from young seedlings (3 months to one year old) or
epicormic shoots (4 to 5 meters high and less than 2 years old) from 30 year
old trees (57). However, cuttings from regular shoots of 4 to 5 year old trees
could not be rooted.
Similary with ~ platyphylla, roots could be formed from herbaceous cuttings
from young plants and from epicormic shoots whereas lignified cuttings from
trees one to four years old were unsuccessful (88).
Epicormic shoots develop in most Eucalyptus species following felling, gird-
ling and after damage due to fire or insect grazing. Many Eucalyptus species,
have the ability to produce very large numbers of basal epicormic shoots be-
cause of the persistence of axillary meristems (26, 74). However, the use of
basal epicormic shoots as cuttings does not provide a universal method of
 155

vegetative propagation of adult Eucalyptus since several important species do

not develop epicormic shoots readily from the base of the stem (71). These are
~ regnans, ~ gigantea (now ~ delegatensis), ~ fraxinoides, ~ nitens and ~

astringens (74). In his 1980 review on the vegetative propagation of Eucalyp-

tus, Hartney gives a list of 78 species of Eucalyptus which have been reported
to form roots on stem cuttings taken from seedlings and/or basal epicormic
shoots (71).
It has also been reported (16) that if trees are pruned to form low hedges,
its cuttings can be rooted. For example, ~ grandis and E. camaldulensis hedges
over 4 years old can produce more than 60 rooted cuttings per hedged plant per
year (71). Another source of tissue for vegetative propagation are lignotubers,
which are swellings in the axils of the cotyledons and first few nodes and
contain numerous buds and meristematic tissues (22). Two year old seedlings of
E. tereticornis have been propagated using pieces of lignotuber tissue (8).

3. TISSUE CULTURE
Callus formation in tissue culture of Eucalyptus has been reported in a
number of species (Table 1). The different plant parts that have been shown to
develop callus include seeds, hypocotyls, seedling roots, stem segments, pet-
ioles, leaf blades, apical shoots, lignotubers, anthers, bark explants and
pollen grains (67).
In early studies (75, 115), coconut milk was found to be essential in the
culture medium to sustain growth of Eucalyptus callus. However, it was shown
(39) that coconut milk was not necessary and that ~ bancroftii callus can grow
vigorously on a completely defined medium and ~ grandis, ~ laevopinea, E.
melliodora and E. nicholli on a medium with casein hydrolysate.
Callus from adult ~ grandis and E. urnigera was grown (49) on a defined
medium with neither coconut milk nor casein hydrolysate. A black exudate arose
in many callus cultures and caused problems (39, 51, 67, 75).
Sussex (115), using single cell cultures of E. camaldulensis that had passed
through 36 passages during three years, and Piton (104), using callus cultures
of E. camaldulensis initiated 10 years previously, both showed that the ploidy
level (2n=22 chromosomes) in this species did not change during prolonged
periods of culture and subculture. However, it was also found that although
mitoses were normal until metaphase, there were often anomalies during anaphase
and telophase in the young dividing cells in the external layers of the callus
(104).
156

Table 1. Eucalyptus species used in callus cultures (Goncalves et al (67.

(Nomemclature follows Chippendale (23.

Species Author

Eucalyptus alba Reinw. ex. B1. Kitahara and Ca1das (77); Goncalves (66).
E. bancroftii (Maid.) Maid. de Fossard (39); de Fossard et al (45); Lee
and de Fossard (81).
E. cama1dulensis Delnh. Jacquiot (75); Sussex (115); Piton (104);
Goncalves (66).
E. citriodora Hook. Aneja and Atal (2); Lakshima Sita (79);
Lakshima Sita and Vai1yanathan (80).
E. cladocalyx F. Muell. Jacquiot (75).
~ gomphocephala DC. Jacquiot (75).
E. grandis Hill ex. Maid. Cresswell and de Fossard (29); de Fossard
(39); de Fossard et al (45); Goncalves
(66); Kitahara and Caldas (77).
E. gunnii Hook f. Jacquiot (75).
~ ~inea R.T. Pak . ' de Fossard (39); de Fossard et a1 (45).
~ macu1ata Hook. Goncalves et al (67).
~ melliodora A. Cunn. ex Schau. de Fossard (39); de Fossard et a1 (45).
~ nichollii Maid. and Plake1y de Fossard et a1 (45).
~ nova-anglica Deane and Maid. Winton (117).
~ ob1iqua L'Herit. Blake (9).
~ polybractea de Fossard (39).
E:- robusta Sm. Goncalves (66).
~ saligna Sm. Goncalves (66).
E:- tereticornis Sm. Jacquiot (75); Goncalves (66).
~ x trabuti (hybrid Marcavi1aca and Monta1di (85).
-- botyroides x camaldulensis)
E. urnigera Hook. f. de Fossard et a1 (45).
~ urophylla S.T. Blake Goncalves et a1 (67).
~ viminalis Labill. Blake (9).

3.1. Organogenesis in callus

Plants have been regenerated from lignotuber callus (2), and from cotyledon
callus of ~ citriodora Hook., and from hypocotyl callus of ~ alba (77). Other
organogenesis reported was the formation of small roots from ~ grandis
seedling callus (39) grown in a medium with 2,4-D in the range of 5 to 80~ M. A
total of 175 combinations of auxin and cytokinins were tested on lignotuber
callus of E. bancroftii and although promising nodular callus was obtained,
there was no organogenesis (81). Regeneration of plants or organs from callus
from selected Eucalyptus has never been reported until 1981 (97) when callus
was induced on embryos and sterile seedlings of selected trees of E. leichow n
1 and thousands of homogenous plant lets developed via embryoids in this callus.
 157

4. ORGAN CULTURE
Contrary to tissue culture where simple cells are cultured, in organ culture
differentiated tissues such as leaves, stems and roots are placed in a control-
led system of nutrients and environment. Roots and/or buds are induced on the
explant either directly or after the formation of a callus. The number of tree
species, including Eucalyptus, which can be propagated by organ culture tech-
niques is increasing (1, 71, 92). These organ culture techniques are now often
being used in preference to the traditional methods of vegetative propagation
of trees because of the very high multiplication rates that are possible (some-
times millions per year) (71).
Preliminary experiments on organ culture of Eucalyptus, were undertaken in
1970 (27, 28). Initially, cultures of apical tips from adult field grown ~

melliodora Cunn were unsuccessful because of difficulties to surface sterilize

the tissues. However, apical explants from aseptic seedlings of E. bancroftii
and apical and nodal explants from aseptic seedlings of ~ grandis and E.
deglupta (28) produced roots in vitro. In the past 10 years, much progress has
been made in the organ culture of Eucalyptus and now this method is successful
wi th seedlings of several species and is being used by AFOCEL to produce clonal
plants for field trials for cold hardiness.
The various organs of Eucalyptus reported to have been cultured in vitro are
leaf, petiole, internode, root and node. Leaf disks, petioles and internodes of
~ grandis (ranging from seedling to 4 years old trees) and leaf disks (from
the crown of 5 year old trees) of E. dalrympleana and ~ macarthurii have pro-
duced roots in vitro (51). The gradient of rooting in ~ grandis organ cultures
was seen to decline from a maximum with leaf disks to petioles to nodes to a
minimum with internodes (49). Buds were never initiated in these tissues.
E. camaldulensis roots have been grown in vitro (5) but there was no bud
initiation. The culture of anthers, pollen and ovaries from E. urnigera was
attempted (Cresswell, 1973; unpublished), but no embryogenesis was obtained.
4.1. Nodes
Nodal explants are, in fact, very small one node cuttings where the shoot
system develops from axillary or accessory buds present in the axil of the leaf
and only roots must be initiated. As with cuttings, the rooting in vitro of
nodes from seedlings and epicormic shoots is much easier than from adult trees.
Plantlets have been developed by the nodal culture of seedling material of the
following Eucalyptus species, E. grandis, ~ gunni, ~ dalrympleana, ~ pauci-
flora (49, 50) and E. ficifolia and ~ grandis (43).
158

Nodes from adult trees are much more difficult to root and to develop into
plantlets. However, success has been obtained from E. grandis plants up to 4
years old, a S year old ~ dalrympleana tree (49, SO, S1), and ~ ficifolia (6,
40). No success with rooting was obtained from nodes of adult ~ urnigera, ~

bridgesiana, ~ gunnii and ~ pauciflora (49) nor from subcultured mUltiple

buds from adult trees of E. polybractea and ~ regnans (43). Nodes may also be
cultured to induce multiple buds which can later be separated and rooted. This
technique, which has proven successful with Sequoia sempervirens (13, 93),
increases the number of clonal plants produced. Using this method on seedlings,
the following Eucalyptus species have been propagated (72); ~ camaldulensis,
~ curtissii, E. ficifolia, ~ grandis, ~ obtusifolia, ~ globulus subspecies
bicostata and E. regnans. Adult E. ficifolia has also been propagated in this
manner (44).
Very high multiplication rates for adult ~ ficifolia, ~ camaldulensis and
E. polybractea have been reported (de Fossard, 1981; pers. corom.) using a
combination of rejuvenation of adult tissues by coppicing and rooting of cop-
pice shoot cuttings. These cuttings which are then pinched to induce basal
branching, provide material for nodal cultures using the bud multiplication
technique.
4.2. Problems encountered in developing the organ culture technique
4.2.1. Obtaining aseptic tissue from field-grown plants. One of the major
reasons that more work has been done with seedling material is that besides the
greater plasticity of the juvenile tissues, it is possible to grow seedlings
aseptically and so eliminate the problems of disinfecting field-grown tissue.
The surfaces of plants carry a microbial flora which, although, unharmful to
the plants, mUltiplies rapidly on the chemically defined culture medium, modi-
fying the composition of the medium and producing conditions that cannot be
repeated. When working with field-grown plants, especially trees, where the
tissues (e.g. stem) can be several years old, it is extremely difficult, if not
impossible in some cases, to find tissue that is in a suitable stage of growth
and yet does not suffer from insect or mechanical damage (even leaf scars are
sites of entry for micro-organisms).
In the species of Eucalyptus tested, i.e. ~ gunnii, E. gomphocephala and E.
camaldulensis, it was reported (7S) that all explants were contaminated by
an endogenous bacterium. Endogenous microbial contamination was a serious
problem with all forest material collected (43). Similarly, complete disin-
fection of tissues taken from grafts of ~ gunnii on ~ gunnii could not be
 159

obtained (49) presumably because the micro-organisms were present within the
tissues.
After surface sterilization by the method of Cresswell and Nitsch (31), fol-
lowed by two to four weeks of culture, bacteria or fungi developed either from
the cut ends of the explants or from the abscission layers of the petiole on
the node when using field-grown plants. To reduce this type of contamination,
surface disinfection (30, 31) was extended by soaking the branches overnight in
running tap water or in a weak solution of calcium hypochlorite prior to the
routine surface sterilization technique. There are many variations of this
technique but success depends on the state of damage of the tissues to be
cultured.
Obtaining aseptic plant tissues threatens to be a major stumbling block for
the adoption of the organ culture technique for routine vegetative propagation
of field-grown plants. Nevertheless, one advantage of the organ culture tech-
nique developed for ~ grandis (31) is that actively growing tissues are most
suitable for root initiation. Tissues which are relatively young should have
suffered less damage than older tissues and thus should be easier to disinfect.
To obtain large numbers of young shoots, glasshouse-grown 18 months old E.
grandis trees were pruned (43). The new growth was protected by covering with
glacine bags, which further reduced the subsequent contamination rate in vitro.
The branches probably should be treated with a fungicide and/or insecticide
before pruning and bagging in order to destroy insect eggs. Although this
reduces contamination of induced coppice shoots on adult trees, the subsequent
effect of the insecticide on the growth of the explant in vitro is unknown and
it still only partially resolves the problem of obtaining aseptic tissues from
adult trees.
Often all cultures or even subcultures suddenly became contaminated by
creamy-white bacteria either as a liquid on the surface of the medium or as a
cloud within the medium. This contamination has been identified at AFOCEL as
Bacillus lentus, which is transmitted by alcohol on the forceps or other
implements used in sterile culture work.
4.2.2. Brown exudate. Various methods of eliminating brown exudate or
preventing its formation by woody species in vitro have been tried but rarely
with complete success (75, 96, 109). The darkening of explants, callus and
medium used in the culture of Eucalyptus tissues has been reported (30, 31, 39,
51, 67, 75).
160

In E. grandis organ cultures, there were found to be two exudates (49). One
exudate, produced as a result of wounding, appeared within one hour of excisiol
of the explants and was aggravated by certain constituents of the culture
medium such as high sucrose, serine, chlorogenic acid, cytokinins (49), a high
boron concentration (67), and light (Table 2). This initial exudate, which
increases with age and woody character of the tissues, led to death of the
cultured explants. The second exudate appeared near the end of the incubation
period and seemed to be a product of dying cells.

Table 2. Effect of light conditions on exudate formation by cultured leaf

disks of E. grandis (49).
Exudate score 0 (no exudate) to ++++ (heavy exudate)

Light conditions Exudate score

Complete darkness o
Diffuse light (1,500 lux) +
Short days 8 hr light/16 hr dark ++
Long days 16 hr light/8 hr dark ++++

With callus of ~ bancroftii and ~ nichollii, it was observed (39) that the
increased amount of exudate corresponded to a decrease in the fresh weight of
the callus produced during the culture period. Similary, it was found (67) that
with ~ grandis anther callus, the callus with the least growth at the end of
the culture period, had the highest polyphenoloxidase (PPO) activity. An analy-
sis of the actual values indicated that the increase in PPO activity was of. the
same magnitude as the increase in fresh weight of the tissues. The presence of
polyvinylpyrrolidone, tyrosine and cysteine were inhibitory to growth and did
not reduce PPO activity (67).
The initial exudate in E. grandis cultures could be reduced and eliminated
(51) in most cases if:
a) healthy tissues of a stem diameter less than 5 mm were chosen and the
surface sterilization process did not damage the tissues;
b) a soaking pretreatment (dissected explants are soaked in sterile dis-
tilled water in the light for three hours before planting) was done and the
cultures were initially incubated in darkness (8 days for E. grandis organ
cultures).
The water in which the ~ grandis tissues were soaked was examined (51) and
it was found that the soak water was inhibitory in the cress seed germination
bios say and in the tomato rooting test. However, after treatment of the soak
 161

water with Polyclar (polyvinylpyrrolidone) to remove phenolic compounds the

exudate was no longer inhibitory. This indicated that the phenols retained by
Polyclar were the inhibitory factor in the exudates. The brown exudate encount-
ered in tissue cultures has often been referred to as "phenols" (3, 75, 96)
without further precision.
The water in which the E. grandis explants were soaked was analyzed for its
phenol content (Table 3).

Table 3. Phenolic content of extracts from leaf disks and nodes of E. grandis
(24 explants/20 ml sterile dist'illed water). Polyphenols expressed as
~g gallic acid equivalents/ml (49).

per mg fresh weight

per explant of tissue

Leaf disk 84 52.5

Node 61 11.7

There were more phenols produced by leaf disks than by nodes possibly
because leaf disks had a greater percentage of damaged tissue from the dissec-
tion.
The types of phenols in the soak water were then analyzed following the
technique of Marigo (87). Since it had been observed (49) that dark prepared
extracts had little or no effect on the cress seed germination bioassay and the
tomato rooting test, both light (8,000 lux) and dark prepared extracts were
analyzed. As can be seen in Table 4, at the time of extraction (To) there were
more phenols and especially flavonoids present in light prepared extracts than
in dark prepared ones.

Table 4. Phenol composition of light and dark prepared extracts of E. grandis

leaf leachates after 0 (To) and 13 (T13) days of storage o~the
extract in light (mg/40 ml, i.e. mg/70 leaf disks) (49).

To (dark) Tl3 To (light) T13

Total phenols (gallic acid

equivalents 2.5 2.14 9.62 8.3 mg
Tannins 0.54 0.58 1.7 2.3 mg
Flavonoids 23 220 200 0 ~g
Simple phenols 1.94 1.34 7.72 6.04 mg
162

These results lead to the question: were the flavonoids the inhibitors? In
the bioassays, there was inhibition for the first 2-6 days before rooting or
germination, depending on the concentration of the leachates. In addition,
observation of the extracts (after removal of the tissues) revealed a change ir
color from yellow to brown with time. The light and dark prepared extracts
were analyzed for their phenol content at To and after storage of the extract
at 25C in the light for 13 days (Table 4). This revealed that in the dark
prepared extracts, there was an increase in flavonoids in the light over the 1~

days period whereas in the light made extracts, where initially the flavonoid
~ontent was high, the flavonoids were completely degraded and disappeared.
Although the procedure of estimating the flavonoid content is limited (only the
flavonoids with 3 hydroxyl groups on the A nucleus like phloroglucinol are
precipitated by formaldehyde (110, it is tempting to postulate an inhibitory
role of flavonoids in the exudates, especially since four rooting inhibitors,
presumed to be derived from phloroglucinol, have been isolated from adult
leaves of ~ grandis (33, 34, 48, 101). It has also been reported (94, 95) that
some inhibitors are decomposed in water.
Flavonoids had little effect on rooting in ~ grandis tissue cultures
because the long time required for root initiation (8 days for cuttings and 2
weeks for nodes in vitro) permitted the flavonoids to be degraded.
These studies (49) have revealed that phenols are present in the leachates
from newly-dissected ~ grandis tissues and that these phenols, particularly
the flavonoid category, are inhibitory to the processes of seed germination and
elongation of tomato roots. In order to obtain tissue and organ cultures of E.
grandis free from initial brown exudate, they must be cultured under conditions
which do not favour phenol synthesis. For example, in the dark or low intensit)
light on a medium devoid of phenol precursers. It was also seen (49) that the
physiological state of the parent plant is extremely important with respect to
exudate formation. Less exudate is produced in culture if the branches are
submitted to a cold pretreatment (5C for several days before dissection) or
the parent plants are grown under short days.
4.2.3. Rooting inhibitors. There are several reports of specific rooting
inhibitors in Eucalyptus. Namely, the three G inhibitors isolated and chemi-
cally identified from adult ~ grandis (32, 34, 94, 95, 99, 100, 113,) and
inhibitor from a crude extract of adult ~ deglupta (37) and more recently,
Grandinol from adult leaves of E. grandis (33).
 163

~ deglupta forms roots readily on cuttings from trees up to one year old
but does not produce roots on tissues from five year old trees. The presence of
an inhibitor in a methanol extract of adult ~ deglupta leaves and stems was
established (37) using the cress seed germination bioassay.
The concentration of rooting inhibitors was found to increase (94, 95) in
successively older leaves of E. grandis. This increase in concentration of
inhibitors was correlated with a decrease in rooting ability of cuttings taken
from successively higher internodes (100). The G inhibitors, which are presumed
to be derived from phloroglucinol, have been found in high concentrations in
adult ~ grandis leaves and in low concentrations in juvenile leaves of ~

grandis and adult leaves of ~ delegatensis, ~ melliodora and Callistemon

lanceolatis (48). The G inhibitors were tested in the mung bean rooting bioas-
say, the cress seed germination bioassay, the Avena straight-growth test and
rooting of ~ deglupta seedling cuttings. However, G was not always a rooting
inhibitor because at between 0.1 and 0.5 mg/1 it replaced auxin in powder dips
and promoted rooting in cuttings of ~ grandis, Lagerstroemia indica and Azalea
mollis. The association between rooting behavior in ~ grandis and bioassay
responses at in vivo concentrations of G, implies that control of rooting may
be regulated by G in ~ grandis. Another possible role for G is indicated by
its apparent ability to reduce wilting during water stress in E. grandis, an
effect that may indicate abscisic acid-like activity (98).
4.2.4. Bud inhibitors. A growth inhibiting substance has been extracted from
mature leaves and bark of ~ obliqua in which apical dominance occurs (10). It
is absent in juvenile tissues of very young seedlings which show weak apical
dominance. Sprouting from lignotubers in decapitated ~ obliqua seedlings is
correlated with a reduction in the level of inhibitory substances. The
re-application of the inhibitory substances, but not abscisic acid, reduced
sprouting.
4.3. Factors affecting root initiation in nodes
Most of the work on rooting of Eucalyptus in vitro has concentrated on vary-
ing the culture medium (40, 74, 81,) and the incubation conditions (40). Little
research has been done on the age and physiological state of the parent plant
and of the tissues to be cultured. These are of great importance because if the
plant tissue is not in a receptive state, then the rooting stimulus cannot be
accepted.
4.3.1. Physiological state of the parent plant. Rooting of Eucalyptus
cuttings and of air layers is best in Spring (21, 24, 53) when there is an
1M

activation of growth. Similarly, the requirements for active growth of the tis-
sues is demonstrated by the fact that cuttings taken from seedlings and young
epicormic shoots are more likely to root than cuttings from adult trees. To
study seasonal effects in rooting of nodes, in vitro cuttings from hedged E.
grandis plants were grown under completely controlled temperature and photoper-
iodic conditions in the Phytotrons at Gif-sur-Yvette. Many different combina-
tions of temperatures and daylengths were tested and as expected, the growth of
the parent plant and the rooting ability of its tissues changed depending on
the conditions. Best rooting of leaf disks, nodes and apices generally occurred
in those of plants which were the most actively growing at the time of dissec-
tion.
It was found that there was a seasonal trend in the number of nodes that
rooted and that the best rooting conditions were not always the same (Table 5).

Table 5. Effect of the environment of the parent plant on the rooting

percentage of nodes taken at two different times. 12 replicates,
incubation 28 D C L.D. (49).

Temperature DC 24/17 17/17 22/12 27/27

Photoperiod L.D. S.D. L.D. S.D.

January 33 33 83 9
April o o 25 50

A high percentage of rooting was obtained initially when the plant was
placed in a new environment because growth of the whole plant was stimulated.
Several months later, there was a decrease in rooting because the plant had
become accustomed to the environmental conditions and zones of metabolic act iv-
ity had been established within the plant. Thus it appeared that the shock of
changing a plant from one set of environmental conditions to another affected
the metabolism of the plant and stimulated rooting. This was also demonstrated
by another experiment (51), in which the rooting of ~ grandis nodes was
determined before transfer of the plants and 3 and 9 weeks after transfer of
the plants to the new environment. Eight plants (regenerated from nodes from 2
plants i.e. two genotypes) were transferred from 24/17 D c L.D. into 4 different
environmental conditions (Table 6); two seedlings were left unchanged. In table
6, there was no significant difference between environmental conditions but
there was a significant increase in rooting of nodes from plants kept between 0
 165

and 9 weeks in the new environment. Nodes of the young seedlings which did not
change environment maintained a steady rate of root initiation. Hence the
active growth stimulated by the change in environment for adult tissues and the
active growth of the seedlings, led to a positive rooting response.

Table 6. Percentage rooting of E. grandis nodes of plants kept for various

times in different temperature/photoperiod regimes (24 replicates)
(49).

22/22C 22/22C 22/12C 24/17C 24/17C

Environment L.D. S.D. L.D. L.D. L.D.
seedlings

Weeks
o 4 0 0 0 58.5
3 12.5 44 8 16.5 66.5
9 29 37.5 21.5 17 64

Similarly, the treatment of whole trees with sprays of gibberellin stimula-

ted their vegetative growth and their leaf disks rooted prolifically in vitro
(49). On the other hand, treatments which inhibited or slowed down plant growth
such as exceptionally cold or hot environmental conditions, prevented rooting
of the explants in vitro (49).
Many examples of increased rooting from actively growing tree tissues can be
found in the literature (12, 17, 55, 60, 61). In organ culture, plantlets have
been developed in vitro from adult ~ ficifolia (42). Initially, the nodes were
cultured and when axillary buds developed, these were excised and rooted. It
was easier to root the actively growing axillary buds than the nodes from the
parent plant.
Another demonstration of the requirement for actively growing tissues for
rooting is the rejuvenation of tissues. In E. camaldulensis, cuttings taken
from rooted cuttings rooted more readily than cuttings taken from the parent
plant (59). Adult tissues were also grafted onto seedling rootstocks to obtain
an activation of growth and a rejuvenation of the tissues (20). The adult Euca-
lyptus scions formed intermediate and even juvenile stage leaves. These sec-
tions were then used as cuttings (Fig. 2).
In organ culture, nodes taken from plants regenerated from nodes in vitro
rooted more readily than nodes taken from the parent plant (49). More recently,
this activation of growth was used (43) to obtain rooted organs in cultures of
166

~ ficifolia, ~ camaldulensis and ~ polybractea. Firstly, the formation of

coppice shoots was stimulated. These were then propagated as cuttings and it
was the actively growing rooted cuttings which provided tissues for the organ
cultures.
Whether in fact, the rejuvenation process increases the rooting ability by
stimulating their metabolic activity or by initiating a specific rooting
stimulus is at present unknown. However, the important fact to be noted is that
for the regeneration of plantlets by organ culture, actively growing tissues
are required.
4.3.2. Position on the parent plant. In seedlings, initially all tissues are
actively growing. It is only later (about 18 months in E. camaldulensis) (59)
that distinct zones of metabolic activity are established and rooting ability
decreases as the tissues become senescent.
The content in endogenous growth substances has been shown to vary with
position of the tissues on the parent plant (69, 76) and with environmental
conditions (114). The endogenous growth substances content of the tissues
varies with ontogenetic and physiological age of the tissues and so each tissue
has a different demand for exogenous growth substances for root initiation to
be induced. This is demonstrated by the work (4) on the auxin requirements for
the rooting of cuttings of ~ camaldulensis.
A mapping was done (88) of the rooting of each node of a 7 month old E.
deglupta tree and of small plants of ~ hybrid urophylla x alba x tereticornis,
which showed that the best area for rooting of cuttings was the top third of
the tree excluding the terminal parts of the branches.
The rooting potential of apices and nodes of ~ grandis was compared (49)
and it was found that apices were less likely to root than nodes. It was also
seen that the rooting potential varied between horizontal and vertical branches
on the tree. Figure 3 represents a mapping of nodes which rooted in vitro when
every node of a 9 month old tree regenerated from a node was cultured. Before
dissec tion the tree was divided into three regions depending on the color of
the leaf on the node: green, green/yellow or red. Although there was little
difference in rooting between green (39.3%) and yellow/green (38.8%), there was
very little rooting from very young nodes with small tender red leaves (12.5%).
The lower nodes on the main stem from which the leaves had already abscised did
not root. The results indicate that the rooting ability of each node is in-
fluenced not only by the ontogenetic level of the node on the parent plant
(effect of vertical node level) but also by the physiological age of the node
 167

node which produce d a root 5

culture contam inated, dead or
with excessi ve exudate (score 4 or
5)
node without root

Fig. 3. Distrib ution of the nodes which formed roots

from a 9 month old E.
grandis tree regene rated from a node. The physio logical
zones as-
determ ined by leaf color are mapped .
168

(effect of horizontal node level), as gauged by the growth of the ,l eaf on the
node. Best rooting occurred in nodes where a functional yellow/green to green
leaf was present and where the axillary bud had not commenced growth before
culture. When nodes with very young leaves less than two-thirds of the final
size, or nodes with senescing leaves or from which the leaves had already
abscised, were cultured roots were rarely initiated under the test conditions.
Choice of replicates should not consist of the last 3 to 4 nodes of each branch
but of higher numbered nodes (apex = 1) and should depend on the length of the
branch, presence of axillary buds and the physiological state of the leaves.

5. USE OF ORGAN CULTURE ON AN INDUSTRIAL BASIS

The rooting of cuttings from the juvenile stage of many species of Eucalyp-
tus is relatively easy and a common practice in Australia (55) and in North
Africa (14) but the propagation of adult trees from cuttings is generally im-
possible. Since 1956, it has been shown that after grafting and especially cut-
ting back, it is possible to root the cuttings taken from the new juvenile
shoots, taken from trees over 30 years old (57). In addition the rooting of
cuttings taken from the first generation of rooted cuttings increased from 8 to
60%. These initial experiments indicated that it could be possible to mass pro-
duce clones of adult Eucalyptus camaldulensis. This was attempted in Tunisia
(59) and has since been obtained in the Congo (21, 88) and in Brasil (73). This
method of industrial production of rooted cuttings from selected adult trees,
revolutionizes the classical means for the genetical improvement of forest
trees (118).
Since 1973, AFOCEL (116) has studied the use of micropropagation in vitro
for the mass production of clones of Eucalyptus selected for their cold resis-
tance in France (Fig. 4). The pioneer works (27, 28, 30, 31) on ~ grandis con-
firmed earlier results (59) indicating the possibility of in vitro propagation
of some species of Eucalyptus. This success encouraged the work with young
plants produced from seeds grown in vitro, or juvenile sprouts fr'om stock
plants (56), and with young plants or new shoots from adult scions grafted onto
juvenile seedlings (82). The few plants (Fig. 5) resulting from these trials
were successfully planted in the field in 1975 and this led to an extension of
the work by developing clones from 2 year old trees which were selected for a
natural resistance to cold conditions in the Marvejols area. Here we shall
describe the techniques developed after a year of intensive research which led
to a pilot production of about 25,000 plants per month.
 169

5.1. Selection for cold hardiness

This programme (86) consisted of taking seeds (free pollination) from trees
planted about 1950 (90) which had survived the cold winters (approx -20C) of
1956 and 1963. The seeds were collected mainly from ~ gunnii, ~ dalrympleana,
E. pauciflora and ~ delegatensis as recommended by Professor Pryor in 1976.
The seeds were sown in pots; after reaching the colyledonary stage the seed-
lings were replanted into "Melfert"l culture containers which are particu-
larly suitable for the growth of Eucalyptus (62). During the summer, these
plants were transferred to large plastic boxes with holes and filled with soil.

Fig. 4. Taking scions from frost resistant E. dalrympleana

Fig. 5. Eucalyptus seedling in vitro; ~ dalrympleana

For natural cold trials, the boxes were taken to the site sufficiently early
for the plants to be hardened off before the first cold. After two winters in
the field the most resistant and most vigorous plants were cut back, cuttings
were grafted onto young seedlings and the new growth from this stock was sent
to the laboratory at Etan~on for mass propagation in vitro. Thus in July 1980,
this technique of mass production was done with the new growth of 13 individ-
uals from winter selection in 1978/79 and 1979/80.

1 "Melfert" is the registered name for special containers designed for the
planting out of in vitro produced plantlets.
170

It is important to underline that even though the clones were entering their
3rd year, they were still relatively juvenile because a continual supply of
juvenile sprouts was obtained by the cutting back operation. Trials using parts
of branches taken from the same clones which were not cut back failed but not
from lack of reactivity but because of infection. The tissues from the cut
back plants are the origin of the actual industrial cultures. Thus this justi-
fies the work being done on the stimulation of rejuvenalized growth on scions
of adult trees by spraying with cytokinins (89).
Plants sprayed with solutions of methanol/water with 50 mg/l of benzylamino-
purine produced an abundance of apparently axillary and perhaps adventive buds
whose morphology appears to be promising even for horticultural type cuttings
in situ. These results are in agreement with those recently published (89) for
E. ficifolia and for Pinus pinaster (35, 63).
5.2. Introduction of clones in vitro
The following methods are presently being used at AFOCEL. As soon as the
branches are taken from the mother plant, the cut ends are sealed with paraffin
and dipped for 8 minutes in filtered calcium hypochlorite solution. Then the
branches are rinsed two times in sterile water and cut into segments consisting
of one node and 1/3rd of each of 2 leaves. Each cutting is planted upright in
an agar medium in a 25 x 150 mm tube with a cellulose stopper. The culture
medium used (medium I) is that of Murashige and Skoog (1962) modified by a
reduction of the calcium by one third. There is a high cytokinin/auxin balance,
BAP 1 mg/l, NAA 10-2 mg/l. In order to avoid browning of the tissues and
the medium, the tissues must be excised and immediately placed in the dark in a
culture room at 25C (50). If the plants have been grown in a glasshouse and
sprayed with the fungicide benomyl 24 to 48 hours before the branches are
taken, then less than 40% of the cuttings become contaminated in vitro.
After 8 days darkness, the cultures are transferred to a culture room with
photo-thermoperiod of 16 hours light (2 x 40 W Sylvania Gro-Lux tubes 30 cm
from the cultures) at 25C and 8 hours darkness at 20C.
After 2 or 3 weeks on medium I, axillary and proventive buds multiply and
develop. Generally, the proventive buds give the best results in the multipli-
cation stages. These buds are separated and transplanted as soon as possible
before the development of suffocating callus from the abscission layers on the
petiole.
5.3. Multiplication of shoots in vitro
Cytokinins were not used in the medium for the propagation of very young
 171

seedlings (50, 82). Multiplication resulted from the repeated elongation and
division of the axillary buds. The technique adopted for 1 to 2 year old clones
selected for cold hardiness resembles the one used by de Fossard et al (44).
Shoots isolated from the primary cultures are transferred either in the first,
third or fourth subculture depending on the clone, onto the multiplication
medium (medium II). This differs from medium I by the vitamins (44) and by a
reduction of BAP from 1 mg/l to 0.1 mg/l with NAA at 10- 2 mg/l in both
media.
If the multiplication is continued on medium I, the tissues very rapidly
become transparent with fragile leaves and stems. Multiplication on medium II
results in intense axillary budding. The leaves become small and thick and have
a cotyledonary morphology and a purple colored lower surface. The very short
internodes thicken and bunches of buds grow in all directions even within the
agar itself. Reduction of the BAP prescribed for medium II, leads to a reduc-
tion in the multiplication rate but allows a stability of the physiological
state of the stock in relation to the rhythm of the subcultures. With monthly
subcultures and stabilized climatic conditions, a multiplication rate of more
than ten per month can be obtained. Contrary to the method of other workers
(50, 82) which requires a careful dissection of the small shoots into nodes, in
this method the clumps of buds are simply divided into ten subcolonies. Further
dissection of these colonies easily produces a propagation rate of more than 30
per culture (Fig. 6).
5.4. Elongation of the shoots
To stimulate elongation of the shoots and to obtain a favourable leaf mor-
phology, two factors, activated charcoal and gibberellic acid were tested, at
first separately, then together. Gibberellic acid in the range 0.01 mg/l to 10
mg/l was added to the medium II before autoclaving. All the concentrations
higher than 1 mg/l lead to a considerable elongation of the internodes of the
transferred shoots. Growth of 30 to 40 mm in one week was not rare, but the
elongated shoots had a undesired morphology; the round leaves became longer and
pointed and their dimensions were reduced from the base to the apex of the
stem. At the same time, the internodes increased from 1 mm to more than 15 rom.
These phenomena occurred in less than 15 days, after this time the apex of the
shoots died and the leaves became fragile and fell at the slightest shock. If
transplanted earlier, the cultures can be rooted and placed again on medium II
but death occurs in 90% of the cases. However, media containing less than 0 .5
mg/l (with an optimum at 0.1 mg/l) gibberellin, though causing less elongation,
172

produced healthier tissues, and better rooting and planting stock.

At the same time the effect of activated charcoal, added to medium II, on
the elongation growth of buds from subcultures on medium II was studied. Adding
1 g/l of activated charcoal to medium II favoured the elongation of one of the
buds of the subculture. During the elongation, the leaf morphology and color
evolved towards that of cuttings of young seedlings obtained in vitro (SO, 82).
This elongation, unfortunately, was relatively small and slow, and since only
one bud grew, it was too low to be of practical value.
Then gibberellin and activated charcoal were tested together. As a result of
factorial trials with diverse concentrations of gibberellin and activated
charcoal added to medium II before autoclaving, medium III, called elongation
medium was developed. This medium consists of the elements of medium II plus
BAP 0.1 mg/1, NAA 10- 2 mg/1 with 1 mg/1 gibberellin and 15 gm/1 activated
charcoal. On this medium 3 to 4 stems per colony of buds grow sufficiently
after 15 to 20 days to give cuttings with large well pigmented leaves and which
are apt to root (Fig. 7).

Fig. 6. Colony of buds in vitro; ~ gunn11

Fig. 7. Elongated axillary bud ready for excision and rooting; ~ gunnii
 173

5.5. Rooting of shoots

The shoots thus obtained are transferred to a Knop medium (78) with 1 mg/1
lBA. The cultures are placed in the dark at 20C for 7 days then returned to
the "multiplication" culture room. Roots then appear in less than one week. As
soon as the root tips appear, the plants must be planted out (Fig. 8).

Fig. 8. Rooted axillary bud; ~ gunnii

5.6. Transfer of plants to soil

The shoots with root initials at their base are placed in specially adapted
"Melfert" containers. These consist of a non-woven sack containing a substrate
of 70% pine bark, 25% sphagnum turf, 5% lignite ashes and 4 g/l osmocote 12/14
month fertilizer. The sack is a 25 x 10 cm (rectangle) which is rolled around
the base of the cutting to form a 300 cm 3 cylinder. The roots form and grow
freely in this cylinder and do not form spirals as is common with the majority
of classical horticultural containers. When the rooted shoot is transferred
from the test tube to a Melfert container, the agar is easily removed because
the roots are not long. However, the Melfert containers must be moistened with
warm water to remove sugar and to reduce thermic stress during transfer of the
shoots. They are then placed directly in trays on irrigation sheets in a con-
trolled climate glasshouse at 20-25C. For the first week, 100% humidity is
maintained by a transparent plastic sheet placed over the trays. This technique
gives over 90% survival. Plant growth is vigorous and it is impossible to dis-
tinguish a plant from in vitro culture from a classically produced seedling
(Fig. 9).
174

Fig. 9. Explants rooted in vitro and transferred to the field; ~ gunnii

6. CONCLUSION
The described method permits a maximum multiplication rate of 30 buds per
culture if the buds are carefully dissected at the end of the multiplication
phase. In practice, this rate is reduced to 10 per month by a quicker random
dissection of the colonies of buds. This is generally sufficient to provide
enough plant material. Cold storage did not stimulate the multiplication rate;
there was no noticeable change in reactions of the buds after cold storage for
6 months.
The culture of buds on a slowly agitated liquid medium has also been tested.
Eventually such cultures could lead to multiplication of shoots of Eucalyptus
clones in chemostats on an industrial scale (15). Dissected shoots could then
be elongated on an agar medium containing gibberellin and activated charcoal in
order to eliminate the unfavourable after effects of the strong concentrations
of cytokinins used in phases 1 and 2.
For sanitary reasons, all operations were done in individual 25 mm test
tubes. However, the rooting phase which lasts only 2 weeks can be done more
economically in 500 ml bottles. Present research suggests that by using special
Table 7. Expenses of the present techniques of in vitro mass production of Eucalyptus

Number of explants treated per 8 hours working at the different conditions

Type of operation Transplanted Preparation Preparation Placed in con- Culture in Yield Cost
under sterile of vessels and distribu- tainers and greenhouse per of
conditions tion of media planted out and harden- plant material
ing off

Multiplication 1500 explants 1500 tubes 1500 tubes 0.95 0.20

(for 3000 (for 3000
explants) explants)

Elongation 1500 explants 1500 tubes 1500 tubes 0.90 0.20

(for 3000 (for 3000
explants) explants)

Rooting 2000 explants 400 (pots) 400 (pots) 0.95 0.05

in bottles for 4000 for 4000
shoots shoots

Acclimatization 2000 contain- 0.85 0.50

ers and shoots
Cultures 10 000 0.95 0.05

- .J
u.
-
176

techniques rooting may be obtained in the elongation medium.

We hope to simplify the production chain to increase productivity in the
future. The economical aspects of this production cannot yet be evaluated pre-
cisely, but the first productivity results in Table 7 give a good indication.
Besides paying off the laboratory and glasshouse equipment, the price per
plant produced is 1 F excluding the cost of labor. If the daily costs are 200
F, then the basic cost of the in vitro plant is about 3 F. This is twice the
price of a seedling grown under comparable conditions. Under such conditions,
the genetic advantage gained by the cloning must be well established for the
method to be practicable. Results obtained in the Congo and Brasil, show that
by using hybrid cuttings, it has been possible to more than double the produc-
tivity of afforestation.
In France, if the cloning permitted the rapid setup of high production tim-
bering, the effect of a relatively high cost of producing clones would be
negligeable if the planting density would be of the order of 1,000 trees per
hectare. For higher densities, the economic interest could be annuled. It is
possible that a follow up with classical cuttings may justify a limited
production of mother plants i .n vitro in the laboratory, the mass production of
plants for re-afforestation being assured by industrial cuttings as in Pointe
Noire or Aractuz (73, 88).
Finally, it was recently reported (97) that embryogenic callus can be easily
obtained. The isolation and transfer to soil of young regenerated plants from
such callus would open new horizons for the mass production as well as the pro-
duction of new varieties, including more frost hardy ones, of Eucalyptus.

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182

7. VEGETATIVE PROPAGATION OF PALM TREES

J.F.REYNOLDS

1. INTRODUCTION
The use of products produced from palms has dramatically
increased. The following chapter will deal with the current
world utilization of palms as a natural resource and the
problems associated with their development. Unfortunately,
traditional breeding methods have not been adequate to fully
expand the potential use of palm products. Palms, as with
most woody species, are difficult to breed largely due to
the time it takes to evaluate test crosses. Plant cell
organ and tissue culture procedures may be a key to speed
breeding programs, and introduction of new hybrids or varieties,
and may aid in the study of palm diseases. In addition,
early in vitro response of palm tissue may be correlated to
increased vigor or heterosis of hybrids, reducing the time
necessary for cross evaluation.
Tissue culture studies of palms are uncommon due to the
difficulties in obtaining and culturing palm tissues. In
vitro studies using Coconut palm tissue were first initiated
in the early 1950's (12). Since this study few new species
have been examined using in vitro systems. Significant
advances in the regeneration of palm tissue in vitro have
only been observed in the past few years (45), (57), (58),
(59)
The following will review most tissue culture studies
of palms and illustrate the difficulties associated with
their culture and progress toward a commercial use of in
vitro techniques.
 183

2. VALUE or ;I?AIJ1S AND PROBLEMS ASSOCIATED WITH THEIR

DEVELOPMENT

2.1. Sources of nutrition

There are presently known about 230 genera and 2,650
species of palms. A great majority of these palms are
utilized as ornamentals, however, 3 species of palms carry
an international economic impact, Elaeis guineensis Jacq.,
the oil palm, Cocos nucifera L., the coconut palm, Phoenix
dactylifera L., the date palm. The former two species are
most important as a source of edible oil, the latter is
used directly as a source of carbohydrate. Other foods
derived from palms include sago starch, sugar, toddy, and
heart-of-palm salad.
2.1.1. Source of edible oils. Plants have always sup-
plied man with a source of edible and industrial oils. Some
of the families supplying oils include: Asteraceae,
Euphorbiacae, Papaveraceae, Sapindaceae, Simaroubaceae and
Leguminoseae (6). The palm family, Palmae (Arecaceae),
may be considered as a major source of edible oils. Some
third world nations rely on the production of palm oil
as a large percentage of export earnings. Nigeria, for
example, earned 32% of its export earnings from palm oil
production between 1.9.49 and .196S (33). Presently, Nigeria
supplies half the world market of palm oil, produced from
the oil palm, Elaeis guineensis Jacq.
The oil palm fruit is a drupe with a thin exocarp, fleshy
mesocarp,and hard endocar.p surrounding an oily endosperm.
The mesocarp and endosperm are both used as an oil source.
Extraction .and processing the oil involves four steps,
sterilization, maceration, extraction, and clarification.
Harvested fruits are sterilized to inactivate lipolytic
enzymes in the mesocarp and to stop oil oxidation. Fruits
are macerated and the fleshy pulp is extracted by mechanical
presses. The kernel or endosperm of the fruit is also ex-
tracted. However, heavy equipment is needed and often
kernels have to be shipped to Britain where such equipment
184

is located. Oil is clarified by bleaching which removes

pigments that lower oil value. Oil resulting from the fruit
is utilized in a large number of ways and influences many
of us daily. Palm oil is used in the manufacturer of
margarine, cooking oil, ice cream, baked goods, and may-
onnaise. Industrial uses include manufacturing of soap,
candles, tin plate, detergents, glycerine, precision instru-
ments, and jet engine lubricants. The residue from the
extracted kernel, or palm kernel cake, is a good source of
protein, for man and livestock.
The future may see palm oil as a source of energy,
partially replacing petroleum, and petroleum based products.
2.1.2. Source of carbohydrate. Coconut in addition to
being an oil source, is used as a source of carbohydrate.
Copra is a product made from dried coconut endosperm and
is familiar to most in preparation of desserts and sweets.
Liquid coconut endosperm or "milk" is sold on the street in
many tropical countries. A delicacy more common to tropical
areas especially southeast Asia, is Makapuno. Makapuno is
a type of coconut completely filled with a gelatinous endo-
sperm and is highly valued as a culinary delight.
P. dactylifera L. fruits, commonly known as dates, are
a food known around the world and have been a dietary staple
in middle-eastern countries for thousands of years. There
are some 75 varieties of dates varying in shape, size, color,
and taste. Dates can be eaten fresh, dried, or packed into
a date cake or Akuch. Akuch can be cut and dissolved in
water, making a refreshing drink. Dates can also be dried
to make a type of flour and dough, or pressed producing a
honey.
Date production in the u.S. is limited to Indio,
California. Here, dates are sold fresh locally but are mainly
harvested dried, then rehydrated prior to shipping.
The use of palms as a worldwide food and oil source is
not yet a reality, however, in developing nations they play
a major part in the economics as well as being a source of
 185

nutrition.
2.2. Ornamental use
Palms are important in the ornamental plant ~nQu~try.

Travel through tropical regions of the world . reveals the

beauty palms add to landscapes. Gardens such as Balboa Park,
San Diego, California, Huntington Gardens, San Marino,
California, and Fairchild Tropical Gardens, Miami, . Florida,
are monuments to the. palm family. Tropical areas are not
the only areas displaying the beauty of palms. Living rooms
and greenhouses around the. world contain species such as
Howeia forsteriana Becc. Rhapis excelsa Thunb. Chamaedorea
costaricana Oerst. or C. seifrizii Burret and Phoenix
Roebelenii O'Brien. Ornamental palms are relatively slow
growing and consequently, require much time and effort in
their production. This translates to high cost per plant
for neighborhood nurseries. Some nursery operations base
most of their sales on palms alone and have been known to
sell $1.5 million of palms per year.
One of the economically important ornamental palms is
the Howeia forsteriana Becc. commonly known as the Kentia
palm or Paradise palm. The Howeia, one of the more beauti-
ful palms, is indiginous to the Lord Howe .Island, a 5 by
7 mile island off the coast of Australia. It is the palm
seen as background in everything from Marx Brothers movies,
to Pres.idential addresses. Approximately 5 to 8 years are
required from sowing Howeia seed to sale of the finished
product. Groups of 3 to 6 palms are sold in single pots
for a price of $30 to $215. Some of the larger specimens
or some of the more rare species, such as the variegated
Rhapis, are worth several thousands of dollars.
2.3. Present methods of cultivation and propagation
2.3.1. Coconut palm (Cocos nucifera L.) The coconut palm
is a single columnar palm, traditionally propagated by
seed, generally cross pollinated and considered to be very
heterozygous. The variability associated with the
heterozygosity can be seen in yield and disease resistance
186

variatien. Yield differences may exceed 40 times within

ene cultivar (50). Heterozygesity is enhanced by the
timing ef fleral maturatien. Female flewers ef a bleeming
tree beceme receptive after the male flewers ef the same
tree step preducing pellen. The next male inflerescence
dees net bleem until the female stage is cempleted. There-
fere, fruit set is dependent en pellen frem an adjacent
tree. The dwarf variety semetimes has an everlap ef
flewering phases resulting in seme self-pellinatien . In-
creased hemezygesity asseciated with this variety is mest
likely explained by selfing.
The element ef time has made breeding pregrams
difficult. After planting, seedlings de net flewer until
the fifth year and 5 to. 10 years are required befere an
accurate evaluatien ef a particular tree can be made.
The time taken to. study enly a few generatiens ef in-
breeding weuld practically exhaust the lifetime ef the in-
vestigating scientists. Seme metheds ef vegetative
prepagatien have been attempted with cecenut. One methed
is threugh air layering ef tall trees (14). This is
achieved by attaching weeden bexes filled with meist sand
er dust to. weunded stems. The newly reeted stem can be
cut belew the bex resulting in a 100 feet tree being re-
duced to. a 10 feet specimen witheut less ef preductien.
This also. makes harvesting and cultivatien easier . The
air layering methed is ef particular interest fer seed
trees which are knewn to. preduce high yielding effspring.
Anether petential methed involves reversal ef palm
spadices frem generative to. vegetative grewth (15).
Vegetative sheets arising frem inflerescences have been
ebserved in many palm species, and are referred to. as
bulbil sheets. Altheugh the cause fer this reversien is
net knewn, it is semetimes asseciated with trauma. One
repert neted that a destreyed terminal sheet resulted in
spadicies reverting to. vegetative sheets (15) . Observatiens
have also. been made en reversien ef individual flewers to.
 187

vegetative shoots (15). If all flowers could be transformed

to vegetative shoots, thousands of clonal shoots could be
produced per year.
A final method for yielding clonal coconut palms has
been the branching of the single shoot. Trees damaged by
lightening have been observed to branch on several occasions
(13). Seedlings split with a blade have resulted in 3 to
4 shoots (15). The origin of these shoots has been studied
by Balaga (5) and were shown to be adventitious in nature,
not axillary, originating from points of injury on abaxial
leaf surfaces near the apical dome,. and near cut areas of
older leaves.
2.3.2. Date palm (Phoenix dactyliferaL,) There .is
evidence for cultivation of date palms in the protohistoric
near east as early as 4000 B.C. (65). References to date
palms have been observed in Mesopotamian wall paintings and
sculptures, as well as Egyptian tombs. The wild stock for
the domesticated variety was thought to originate in the
southern near east. However, there are many other Phoenix
species which freely hybridize with the domesticated
variety (65) . These are distributed in north Africa, Arabia,
and southern Asia. Some examples are P. reclinata Jacq.
of east Africa, and P. Atlataca Chev. of northwest Africa.
Phoenix dactylifera L. is a columnar palm reaching the
height of 80 or more feet. It has the ability to form shoots
from axillary buds commonly referred to as offshoots or
suckers, thereby enabling some varieties to be clonally
propagated. Problems associated with such a propagation
technique include potential distribution of insects and
disease, transport, maintenance and inspection of the off-
shoots, which can weigh as much as 40 lbs. Rapid propaga-
tion of desirable cultivars is further hampered due to the
production of only one or two offshoots over the life of
the palm.
Seed propagation of date is possible but not practical
for several reasons. P. dactylifera L. is dioecious and
188

completely heterozygous in both male and female parents.

There is much segregation in populations derived from seeds,
and varieties can not be maintained. Seedling populations
would also have a superfluous number of staminate trees,
which cannot be recognized until flowering. In addition,
seedlings do not start to flower for 12-15 years while
clonal offshoots are capable of flowering in 5-6 years.
Trees do not bear a full crop until the age of 30 and gener-
ally slow production after 80 to 90 years, although some
have been known to be productive for 150 years. As with
the coconut, air layering techniques have occasionally been
used to rejuvenate some desirable trees.
Good fruit set in date palms requires artificial fertil-
ization of pistillate trees. In addition, a metaxenic effect
has been identified in date palm, necessitating specific
trees for pollen source, as well as a seed parent.
2.3.3. Oil palm (Elaeis gl\:i,nees.i.sJa,cq.) The o.i.l. palm,
like the coconut palm, has similar problems associated with
its propagation. Vegetative propagation has not been
possible except in rare occasions where inflorescences or
flowers have reverted to vegetative shoots. Some attempts
have been made to revert inflorescences to vegetative shoots
by the addition of GA3_ to flower axis (26), however, this
was not successful. Propagation of oil palm has been
strictly through seed and has many problems. Germination
of the seed is a major effort. Oil palm seed has a
dormancy which increases for two months after harvest (61)
and requires special methods to overcome. Seeds are first
rehydrated by water soaking for .7 days, then placed in a
germinator at 39C for 80 days. Subsequently they are
resoaked for 7 days at ambiant temperature and germinated
(34)
Oil palm, like the coconut palm, is very heterozygous
and varies dramatically with respect to yield, disease
resistance, and oil quality. Conventior,cal breeding programs
have been in progress and have made some advances in yield.
 189

A useful hybrid was made by crossing two parental types,

the Dura, a thick shell type, and Pisifera, a shelless type,
resulting in the Tenera hytrid. Tenera fruit has a fleshy
mesocarp which is milled off leaving a thin shelled nut or
kernel. The kernel is mechanically extracted giving oil
high in lauric acid which is desirable for detergent produc-
tion (27). Parental lines of Dura and Pisifera are not
well inbred and the resulting progeny, although greater in
yield, are still variable, and cannot be evaluated for a
minimum of 7 years (54).
Additional problems have arisen due to the evolution
of oil and fat technology. Certain products require a
specific oil make-up such as a low caratine content or
specific triglyceride make-up (27). Traditional breeding
schemes would require a great amount of time and effort to
incorporate such specific oil qualities into the oil palm
fruits.
2.3.4. Ornamental Palms. rropagation o~ ornamental palms
is done mostly by seed. However, many palms, for example
Chameadorea or Rhapis, have rhizomes enabling vegetative
propagation by division of newly produced shoots. Seed pro-
duction has many problems, including reliable seed source.
For example, the Howeia forsteriana Becc. is a very desir-
able ornamental, but is indigenous only to the Lord Howe
Island off the coast of Australia. If one were fortunate
enough to secure the seed, germination would require a year
or more in special germination beds with bottom heat or
charcoal bedding medium. Difficulty in germinating many
other species of ornamental palm seeds is evidenced by the
many methods employed to increase germination percentage
or rate. Bottom heat (43) presoaking (43)scarification (25)
and the addition of growth regulators (32) are a few of the
methods employed. Embryo culture is another method used (24).
190

3. SOLVING PROBLEMS WITH TISSUE CULTURE - CURRENT

STATUS OF RESEARCH
The previous section discussed methods for propagation
of three economically important oil palms as well as some
ornamentals. It becomes obvious that there are many prob-
lems associated with conventional propagation methods.
utilization of tissue culture procedures for propagation
may circumvent many of these problems. Tissue culture
investigations of palms have been limited to three species:
Cocos nucifera L., the coconut palmi Phoenix dactylifera L.,
the date palmi and Elaeis guineensis Jacq., the oil palm.
Reynolds and Murashige (45) were the first to use tissue
culture for investigations on regeneration in ornamentals.
They specifically studied Howeia forsteriana Becc., and
Chameadorea costaricana Oerst., as well as the date palm,
Phoenix dactylificia L.
3.1. Cocos nucifera L.
An early study by Cutter and Wilson (12) used coconut
palm embryos to test Caplin and Steward's (9) coconut milk
factor. They speculated that the factor might have an
important role in the development of the coconut embryo.
They determined that unautoclaved liquid endosperm from
immature coconut fruits was beneficial and that from mature
fruits was inhibitory. The inhibition may have been due
to substances produced by the endosperm which caused
dormancy of the embryo in vivo. Abraham and Thomas (1)
confirmed that unautoclaved coconut milk from young fruit
stimulated coconut germination. In addition, their study
suggested that a standardization of the coconut germination
procedure might be useful in rapid identification of crosses
that manifested hybrid vigor. Abraham and Thomas (1) were
also the first to suggest embryo culture from '11akapuno'
coconut fruits. The fruits of this cultivar, the ovules
of which are completely filled with endosperm, produce
embryos that normally abort. They proposed that embryo
culture would produce trees bearing exclusively 'Makapuno'
fruits. DeGuzman and Del Rosario (16) cultured embryos from
 191

'Makapuno' fruits and were able to attain well-developed

shoots, however, root formation was poor. Ventura et al.
(62) also achieved limited success in the culturing of
'Makapuno' embryos. Both studies encountered the problem
of browning of tissue and medium. However, neither study
mentioned reculturing, even after four months. Balaga and
DeGuzman (5) were more successful in germinating 'Makapuno'
embryos. They planted embryos with emerging shoots in
liquid media to facilitate root growth. They suggested
that the brown exudate produced by the excised embryo
inhibited root growth, and that this inhibitor might be
removed more rapidly from;the embryo in liquid media.
Germinated 'Makapuno' embryos produced abnormally small
adventitious roots when .recultured in media supplemented
with 8% dextrose and 0/25-1.0 mg/l NAA (48). Roots were
produced from new mer/stems that formed from cells asso-
ciated with procambial strands.
In later studies other explants of the coconut were
cultured with the hope of producing tissue or cell cultures
that could be used in morphogenetic studies.
Balaga (4) observed occasional branching of adult coco-
nut palms; this initiated investigations regarding the origin
of the branches. Adult coconut palms generally produce
branches as a result of injury or disease. The origin of
these newly produced shoots was generally believed to be
axillary. However, using histological methods, Balaga ob-
served that bisected shoot apices of coconut formed meristems
on the abaxial surfaces of leaf primordia or young leaves;
additional buds were formed in areas lateral to the apical
dome, or on older leaves near the injury site. Therefore,
this study presented evidence against axillary branching
in coconut, and also disclosed the potential for vegetative
propagation of coconut.
Eeuwens (17), in attempting to culture excised coconut
tissue, developed a salt formulation that enabled the culture
of coconut inflorescence segments. This salt formulation
192

was claimed to be superior to that of Murashige and Skoog

(31). He noted that microelements, particularly iodine,
were limiting in the MS (Murashige and Skoog) medium. Or-
ganic and hormonal requirements of coconu.t and date palm
tissues were also investigated by Eeuwens and Blake (19)
and Eeuwens (18). During the investigations, shoots and
roots developed from inflorescences of coconut palms, and
callus was produced from root, stern and inflorescence
sections. Fisher and Tsai (21) were able to culture coconut
embryos and noted that inclusion of charcoal in the nutrient
medium promoted embryo growth. They also reported that
callus arose from the endosperm. The callus was maintained
four years, and occasionally produced finger-like projections
that lacked meristematic or vascular tissue. The chromosome
number of the callus was 8 (2n= 32). Attempts were being
directed toward obtaining the normal diploid chromosome
number to possibly increase changes in organized development.
3.2. Phoenix dactylifera L.
Vegetative propagation of desirable cultivars of P.
dactylifera L. has been accomplished slowly by the very
cumbersome method of rooting offshoots (axillary shoots at
the base of a mature tree).
Schroeder (49), using various tissues excised from off-
shoots of the date palm, was able to produce a few viable
cultures. He obtained callus in one instance, and was able
to maintain the tissue in vitro for three years. Root for-
mation was reported in the callus culture. Embryo culture
was also accomplished by Schroeder. Reuveni and Licien-
Kipnis (44) succeeded in establishing date palm tissue
cultures by using explants of embryos obtained by germinating
seeds in vitro. Cotyledonary sheath sections obtained from
the embryos generated callus and adventitious roots, but not
shoots. Smith (53) claimed to have produced plants from
root tips excised from seedlings obtained in vitro. It is
probable that Smith's root tip was in reality the cotyle-
donary sheath, which normally contains the shoot-root axis
 193

but resembles root ti,ps. Anul\ar .andBenbad;ls (2) were able

to produce callus in cotyledonary sheath sections taken from
zygotic embryos germinated in vitro. The callus formed shoots,
roots and, occasionally, inflorescences.
Recently, Reynolds and Murashige (45) demonstrated that
germ pore containing ovule sections of date palm were able
to produce callus on a medium with 100 mg/l 2,4-0 and 0.3 %
charcoal. Subculture of this callus to a medium without
2,4-0 resulted in the formation of somatic embryos. A
histological study demonstrated that the development of the
somatic embryos resembled zygotic embryo development. They
attributed their success to the immaturity of the original
embryo explants. The most responsive explants were parts
of 2-3 month old ovules which presumptively contained a
developing embryo.
Reynolds and Murashige (45) also reported on culture of
embryos or inflorescence tissue from ornamental palms, in-
cluding Howeia forsteriana .Becc. and Chamaedorea costaricana
Oerst. They observed somatic embryogenesis from an embryo
derived callus of these species, however, not as prolific
as in that of date palm, Phoenix dactylifera L. Plant
regeneration was also observed from inflorescence explants
of c. costaricana Oerst. (46). Plants were shown to regen-
erate by two distinct methods. Inflorescence explants cul-
tured on 100 mg/l 2,4-0 produced callus and somatic embryos.
Subculture of this tissue to medium without 2,4-0 resulted
in development of the embryos to plants. Shoots were also
observed to form directly from the inflorescence explants,
presumably by reversion of generative meristems to vege-
tative shoots. These shoots continued to multiply at a
slow rate by enhanced axillary branching.
Tisserat (57), (58) and Tisserat and OeMason (59) re-
peated and confirmed Reynolds' and Murashige's observations
of embryogenesis from both embryo and clonal date palm
tissue explants.
194

3.3 Elaeis guineensis Jacq.

The oil palm has been studied more extensively in tissue
culture than the coconut or the date palm . Substantial work
has dealt with cultures of oil palm embryos. The effort was
intended to gain knowledge of morphogenesis that may be
applicable to asexually derived plants. The oil palm embryo
is a small conical structure embedded in the peripheral re-
gion of the endosperm. During germination the cotyledon, or
haustorium, elongates and penetrates the endosperm. Nutri-
ents from the endosperm feed the petiole, which enlarges to
produce plumule and radicle simultaneously. Embryonic de-
velopment in vitro differs from that in vivo by a haustorium
that is smooth and unfolded and by usually producing either
plumule or radicle, rarely both. Petioles excised without
haustorium will not develop; the haustorium by itself will
enlarge, become green and accumulate starch. The haustorium
normally does not turn green, but may transfer precursors of
chlorophyll and organic substances to the developing petiole
(37). Petioles failed to develop in embryos of seeds that
were too dry (37). Rabechault and Plantefol (41) also ob-
served that embryos did not develop in seeds containing less
than 18% moisture. Seeds hydrated to a 20-22% moisture
content produced maximum embryo growth. Rabechault and Ahee
(35) reported that embryos of large seeds grew more vigor-
ously that those of small seeds and that embryos excised
from seeds stored more than six months developed more slowly
than those from freshly harvested seeds.
Oil palm seeds have been observed to manifest dormancy
during the first two months after harvest. This dormancy
could be overcome by exposing fruits to a 40C temperature
in a humid environment for 40 days. (61). The time required
for treatment at the high temperature was reduced by longer
storage of the fruits, up to 12 months, with a slight release
from dormancy over the 2 to 12 month storage period. Rabechault,
Guenin and Ahee (38), using excised embryos, observed that
during the first two months following harvest, embryos from
 195

dormant seeds developed much more slowly in vitro than those

of nondormant seeds. After 67 days in storage, embryos from
all seeds developed, indicating release from dormancy, even
though the moisture content was only 10%. The researchers
concluded that dormancy was independent of water content of
fruits, opposing the views of Rabechault and Plantefol (41).
Rabechault, Guenin and Ahee (39) reported that carbo-
hydrate supply was not as important as salt provision during
the initial stages of embryo culture. The salt formulation
of Heller (22) was reported to be superior to that of
Hoagland and Arnon (23), Knop (29), Randolph and Cox (43),
Rijven (47) and White (63). Rabechault, Guenin and Ahee (40)
also observed that coconut milk was beneficial only to
embryos excised ~rom ~ged seeds. The maximum effect of coconut
milk was seen among embryos from seeds 45-60 days after
harvest.
The addition of GA3 did not influence the development
of haustorium of excised oil palm embryos (7). However,
appearance of the plumule was accelerated by concentrations
above 1 x 10-6M and root formation was suppressed, partic-
ularly in an agar medium. Auxins and cytokinins had marked
effects. lAA at 1 x 10-7M suppressed plumule formation,
but stimulated rhizogenesis. NAA increased haustorium size
and suppressed shoot and root formation . 2,4-0 showed
effects similar to NAA; however, after 30 days in culture
nodules were reported from unorganized cells along the
fascicular procambium. Kinetin promoted browning of the
embryos to a greater degree than auxins and caused elongation
of the haustorium.
Subsequent work on embryo culture dealt mainly with re-
generation of embryonic callus tissue. Smith and Jones (54)
reported that callus cultures were easily established from
embryo explants of oil palm, and that certain nutrient
formulations supported growth of callus from seedling tissue.
A wide variety of media and environmental factors had little
effect on tissue growth, and the only critical nutrient
196

component was auxin.

Embryos cultured from immature fruits of oil palm formed
nodes near the cotyledonary petiole (36). Following trans-
fer to a medium containing IAA and ascorbic acid, the nodes
produced roots or turned green when exposed to 9-hour daily
illumination, but did not yield shoots. The nodes were
suggested to be embryoids, inasmuch as they resembled early
adventitious embryo initials of carrot tissue cultures.
Jones (28) observed differentiation of white nodules in
callus that originated in aseptically germinated embryos.
The nodules appeared similar to the embryoids of Rabechault,
Ahee and Guenin (36). They contained proteins and triglyc-
erides as found in zygotic embryos. The nodules elongated
and formed shoots and roots. The plants obtained have been
placed under field culture for further observations (10).
Significant progress has been made by the Unilever
Research Laboratories, Colworth House, Bedford, United
Kingdom (11). Nearly 3,000 palms from tissue culture
representing 32 clones, were planted in test plots in
Malaysia. Some 15 oil palm clones were planted in replicated
field trials with fruits being harvested and evaluated. The
tissue of origin for all clones was seedlings, and was not
selected for yield or oil quality. The uniformity within
the clonal palms was much higher than in seedlings populations,
therefore, the asexual produced palms were thought to be
genetically stable. This has not been the case in certain
species cultured in vitro (51), (56).
Since selected clonal material was not used . in these
tests, no attempts were made to evaluate yield increases.
Nevertheless, the presence of a number of genetically similar
plants would be useful in identifying genetically heritable
traits as opposed to those more influenced by environmental
conditions. It was noted through the use of seedling clones
that fruit characteristics are more heritable than yield of
fruit bunches. Some clones were noted to be precocious for
flower production, which may be associated with higher yields.
The genetically uniform material may also be useful in
 197

general agronomic testing, such as, planting densitity, and

nutritional requirements . The ultimate use of clonal propa-
gation will be the planting of palms produced from selected
superior trees. This has recently been initiated and the
clonal material has been planted in the field trials (11).
The tissue was taken from trees producing 50% more oil than
the average means. The selection was made from trees with
a high oil to bunch ratio, a trait noted to be genetically
controlled, therefore, the clonal material should have yields
similar to the progenitor tree .
There have been relatively few instances where use of
tissue culture theory and strategies for improving food
crops have found practical application. Only a few large
companies with tissue culture programs have used their
technologies for production of new varieties. It is inter-
esting that one of the more recalcitrant species to manip-
ulate in tissue culture is at the doorstep of commercial-
ization . Personal communication with researchers at the
Unilever Company revealed that clonal palms produced via
culture would soon be tested, paving the way for commer-
cialization .

4. PROBLEMS OF PALM TISSUE CULTURE

4.1 . Obtaining explant tissue
Tissue culture of palms is in general a difficult under-
taking. It is often difficult to obtain sufficient explant
tissue to work with. There are few species of palms endog-
enous to the United States. Washingtonia filifera, Linden
and W. robusta Wend L. the California fan palms, and some
Florida species, i.e. cabbage palm, Sabal palmetto (Walt.)
Lodd . ex Schultes, and the Sargent palm, Pseudophoenix
sargentii Wendl. ex Sarg. are some examples. Therefore, all
other explant material has to be imported from tropical
areas, or from greenhouse or Botanical garden stock . The
cost of some ornamentals prohibit their use in many research
efforts. Embryo expla.nts have been used by many, however,
198

even in these cases seeds are hard to obtain or are expensive,

as with the $l.OO/seed Howeia forsteriana Becc.
When mature palms are available for explant tissue, it
is difficult to obtain meristematic tissue other than inflo-
rescence tissue. A typical example is the date palm. Small
undeveloped axillary buds are present in offshoots as well
as in the main stem, and are good sources of explant tissue.
Obtaining only a few axillary buds involves using chain saws,
crowbars, sledge hammers, or machetes. The spikes on the
leaf petioles have been known to pierce boots and legs after
being thrown by chain saws used to cut trees. Fibrous leaf
sheaths encompassing the stems are extremely tough and are
only removed with great difficulty.
4.2. Browning of tissue
A problem associated with culture of palm tissue is the
rapid browning of tissue following excision. Many workers
have reported browning of palm tissues, which was thought
to be caused by phenolic compounds produced by wounded
tissue. Several techniques have been employed to alleviate
tissue browning. Reynolds and Murashige (45) utilized an
antioxidant solution containing 100 mg/liter ascorbic and
150 mg/liter acetic acid to maintain tissue after initial
incision prior to disinfection. They also tested PVP, MW
10,000 and 40,000 but found no beneficial effect from its
use. Other workers, who utilized PVP also found no effect
(58). Activated charcoal has been used by many workers
with some success in culture of palm tissues (45), (57).
Reynolds (46) used activated charcoal in his medium at the
rate of 0.3%. Growth regulators added to the medium were
absorbed by the charcoal necessitating the addition of
100 mg/l 2,4-0 in the medium to induce callus formation.
Tisserat (58) also used high levels of auxin in his medium
(50-100 mg/l NAA) due to the use of 0.3% charcoal.
4.3. Regeneration of adult tissues
In many woody species it has been found that adult
tissue is difficult to culture and regenerate, therefore,
 199

embryonic tissue has been used as explant source on many

occasions (64). This has also been true in the culture of
palm tissues (45) . Most early work showing positive re-
sults utilized embryonic tissues (45). Later it was deter-
minec that techniques established for culture of embryonic
tissue could also be applied to adult tissues (58). Best
success has been achieved by using inflorescence tissue (46).
4.4. Sterility of tissue
Early workers found disinfection of tissues difficult
(44). Later, use of embryonic tissues resulted in a source
of tissue requiring little disinfection. Condition of the
original donor plant was important for successful establish-
ment of sterile tissues. Reynolds, utilizing potted
Chamaedorea costaricana Oerst., established axenic terminal
and axillary bud cultures by first allowing the soil to dry
out, then pretreating the plants with a solution of an
industrial disinfectant (Physan) two days prior to bud ex-
cision. This procedure along with disinfection of buds
using vacuum and agitation resulted in axenic cultures .
Tisserat used a final rinse of sodium hypochlorite prior
to culture of date palm shoot tips (58).
4.5. Regeneration fre quency
The frequency of regeneration in, Falm tissues has not
been discussed in any detail in the current literature.
Reports emphasized only that organogenesis has been achieved
in the cultures. Previous discussion has shown that most
palm tissues possess the ability to form somatic embryos.
Steward (55) estimated that 200 ml of medium could produce
as many as 10 5 plants in suspension cultures. Potential for
high frequency regeneration in palms is possible but most
probably will not be necessary due to the relatively low
numbers of plants required for groves and the long term
service of a particular tree. Enhanced axillary branching
would be an ideal method for low frequency propagation of
palms, however, only suggestions of this method have been
reported (46). Adventitious shoot formation either from
200

callus or direct from explant has not been reported. Rever-

sion of generative meristems to vegetative ones would also
be an ideal method of moderate frequency regeneration.
Coconut and oil palm inflorescences have been observed to
revert to vegetative shoots in vivo (15). Concurrent with
these observations are those of Reynolds (46) who observed
in vitro reversion of C. costaricana Oerst. inflorescence
-- -----
flower buds excised from immature rachillae. Stability of
these plant~ was not studied, however, the simple reversion
of meristems should not result in genetically abnormal
growth.
4.6. Growth rates in vitro
Palms are generally slow growing plants, as is reflected
by the time necessary to germinate seeds and to establish
and regenerate cultures. An early paper by Abraham and
Thomas (1) observed that germinating excised coconut embryos
took 6 months to reach the stage or primary leaf formation.
Jones (27) noted two classes of oil palm callus cultures,
one with a doubling time of 30-40 days, and a more rapid
growing callus with a doubling of 10-20 days. Corley,
Barret and Jones (10) noted a doubling time of oil palm
tissue cultures in the range of 20 days. Reynolds and
Murashige (45) noted that callus from presumptive embryo
explants took 10 weeks to develop a 0.5 cm sized callus.
An additional 6 weeks culture of this callus was needed for
somatic embryo formation. Four weeks additional culture
were required for development of embryos to small seedlings.
Tisserat (37) noted development of embryos from bud callus
after 3-4 months culture on hormone-free media.
4.7. Preservation of tissues
The preservation of desirable selected germ plasm may
be necessary if the original tissue donor is lost. Much
basic work has been done in the area of cryopreservation (3),
and some workers have experimented on cryopreservation of
date palm tissues. Finkel, Ulrich and Tisserat (20) pre-
served callus tissue from date palm embryos in liquid
 201

nitrogen. Three months later callus was rapidly thawed,

placed in culture, and observed to produce shoots and roots,
probably through somatic embryogenesis. This is not the
only effort to maintain clonal germplasm of palms. Other
potential techniques, more labor intensive and with potential
genetically drift, include serial culture, reduced temper-
ature growth, or preservation with reduced 02 tensions (8).

5. FUTURE RESEARCH AND PROSPECTS

5.1. Organogenesis - embryogenesis
Knowledge of factors underlying organogenesis have not
significantly advanced since the observations of Skoog and
Miller (52) on cytokinin auxin regulation or organogenesis.
Basic studies of the physiology, biochemistry, and molecular
biology of organogenesis are progressing. ~ However, studies
have been hampered by lack of an orqanoqenic svstem. without.
surrounding nonorganogenic tissue. Advances in this area will
help in all aspects ot regeneration and will certainly be
applicable to palm tissue cultures. Our basic knowledge of
factors underlying somatic embryogenesis are similar to that
of organogenesis. There are many reviews on the subject of
embryogenesis (60). In many systems embryogenesis is
achieved by first inducing callus using a high level of
auxin, usually 2,4-0, with subsequent subculture of estab-
lished callus to media lacking auxin. This sequence is also
observed in palm tissue cultures. The mode of action of the
auxin which causes callus to become embryogenic is presently
not known. In addition, methods of somatic embryo synchroni-
zation, and breaking of dormancy need to be determined and
understood for a full application of somatic embryogenesis.
The low number of plants needed for palm propagation will
not require special staging, therefore, regenerated plants
can be used in a breeding program or for direct field
planting. Reports on stability of palms produced through
embryogenesis indicate that for the palm system, embryogen-
esis results in a high percentage of genetically stable
202

plants (11).
5.2. Inflorescence reversion
The physiology of inflorescence reversion from generative
to vegetative meristem would be particularly useful to palm
breeding programs. Reversion of generative meristems have
been noted in vivo (14) and more recently in culture (46).
This phenomenon has also been noted with species of the
Liliaceae (15). In vitro culture systems may be useful in
studying factors influencing this reversion.
5.3. Breeding programs
Tissue culture can be used in a variety of ways to
supplement current breeding programs of food palms. Propa-
gation of Fl hybrids difficult to obtain could be more
easily done. Haploid production via anther culture would
be useful due to the extreme heterozygosity associated with
coconut, date, and oil palms. Embryo culture would be im-
portant in obtaining Makapuno coconut trees, since Makapuno
embryos normally abort. In addition, hybrids with abortive
embryos. due to endosperm abnormality or incompatibility
could be rescued. Proper evaluation of hybrid coconut or oil
palm trees cannot be done until trees have developed fruit.
In these crops such evaluation may take 10 years or more.
Embryo culture may aid in early identification of crosses
with hybrid vigor, assuming that rapid vegetative growth
can be correlated with yield.
5.4. Disease investigations
In ,vitro culture techniques for disease studies of palms
have been reported by Fisher and Tsai (21). These workers
speculated on methods useful in understanding lethal yel-
lowing disease (LY) of coconuts. LY has been affecting
coconut palms in the Carribean since the early 1960's.
Florida was influenced by LY in the early 1970's. The cause
of the disease has been speculated to be a mycoplasm, which
can be suppressed by antibiotics. Transport of the anti-
biotic Oxytetracycline HCl was studied by McCoy (30) with
the aim of controlling LY. The mode of transmission of the
 203

ct~sease ~s not presently known, therefore tissue culture

studies were initiated to aid in these studies. Tissue
culture regenerated uniform plants would provide an ideal
system to study host pathogen interactions. Experimentation
could be done in a relatively small area, and the pathogen
could be cultured in a living tissue. Furthermore, resis-
tant varieties could be in vitro propagated to study the
resistance mechanism and as a practical application, could
replace diseased coconut groves and ornamental species
affected by lethal yellowing in Florida and the Carribean.

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206

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palm inflorescences In Propagation of Higher Plants
through Tissue Culture-Emerging Technologies and
Strategies, Symposium held at University of Tennessee,
Knoxville, 1980. Environmental and Experimental Botany:
(In Press)
47. RIJVEN AHGC 1952 In vitro studies on the embryo of
Capsella Buesa-pastorrs:-Acta Bot Neerl 1,2: 157-200
48 . SAJISE JU, EV DE GUZMAN 1972 Formation of adventitious
roots in coconut makapuno seedlings grown in medium
supplemented with naphthalene acetic acid. Kulikasan,
Philipp J BioI 1: 197-206
49 . SCHROEDER CA 1970 Tissue culture of date shoots and
seedlings. Report of the 47th Annual Date Growers
Institute 47: 25-27
50. SCHWABE WW 1973 The long, slow road to better coconut
palms. Spectrum 103: 9-10
51. SKIRVIN RM 1978 Natural and induced variation in tissue
culture. Euphytica 27: 241-266
52. SKOOG F,CO MILLER 1957 Chemical regeneration of growth
and organ formation in plant tissues cultured in vitro.
Symp Soc Expt BioI XI: 118-131
53. SMITH SN 1974 Vegetative propagation of the date palm
by root tip culture. Bull Dagron Sharienne 1: 67
54. SMITH WK, LH JONES 1970 Plant propagation through cell
culture. Chem Ind 44: 1399
55. STEWARD FC, MO MAPES, PV AMMlRATO 1969 Growth and
morphogenesis in tissue and free cell cultures. In FC
Steward, MO Mapes, PV Ammirato, eds, Plant Physiology:
a treatise, 5b, Analysis of growth: the responses of
cells and tissues in culture, Academic Press, New York,
pp 358
56. THOMAS E, PJ KING, I POTRYKUS 1979 Improvement of crop
plants via sinqle cells in vitro-an assessment. Z
Pflanzenzuechtg 82: 1-30-- -----
57. TISSERAT B 1979 Propagation of date palm (Phoenix
dactylifera L) in vitro. J Exp Bot 119: 1275-
1283
38. TISSERAT B 1979 Tissue culture of the date palm. The
J Hered 70: 211-222
 207

59. TISSERAT B, OA OEMASON 1980 A histological study of

development of adventive embryos in organ cultures of
Phoenix dactylifera L. Ann Bot 46: 465-472
60. TISSERAT B, EB ESAN, T MURASHIGE 1979 Somatic embryo-
genesis in angiosperms. In J Janick, ed, Horticultura
I Review. AVI 8 Publishing Co, Westport, Conn pp 1-78
61. TROUSLOT MF, G GUENIN, H RABECHAULT 1967 Conservation
et dormance des graines d'Elaeis guineensis Jacq.
Oleagineux 22: 295-296
62. VENTURA PF, LC ZUNIGA, JE FIGUEROA, FO LAZO 1966 A
progress report on the development of coconut embryos
in artificial media. Philipp J Plant Ind 31: 81-85
63. WHITE PR 1943 A handbook of plant tissue culture, The
Jaques Cattell Press, Lancaster, Penn, pp 277
64. WINTON LL 1978 Morphogenesis in Clonal Propagation of
Woody Plants In TA Thorpe, ed, Frontiers of Plant Tissue
Culture. Proceedings of the 4th International Congress
of Plant Tissue and Cell Culture, Calgary, Alberta,
Canada pp 419-426
65. ZOHARY 0, P SPIEGEL-ROY 1975 Beginnings of fruit growing
in the Old World. Science 187: 319-327
208

8. PHYTOPATHOLOGY AND TISSUE CULTURE ALLIANCES

H.V. AMERSON AND R.L. MOTT

1. INTRODUCTION
This volume is primarily devoted to tissue culture as
related to forestry. Application of tissue culture to forest
species has only a short history, thus little information is
available on its use for in vitro forest pathology. However, the
potentials of in vitro cultures for the study of phytopathology
have been demonstrated with horticultural and agricultural plants
which have a rich tissue culture tradition. It is worth our time
to become aware of these potentials and technologies because the
in vitro approach may find its best expression in forestry where
the study of pathology is hampered by tree size, rugged terrain
and variable environment over the long forest life.
It is our intention in this chapter to highlight, without
exhaustive review, the tissue culture-plant pathology studies
involving viruses, bacteria, fungi, nematodes, and insects.
Where possible, reference will be made to research dealing with
tree species. We will not expound on the work done in
agriculture and horticulture for this is discussed in detail in
recent reviews (43, 44, 45, 47). These reviews are excellent
works and the major points and examples used in this chapter are
further elaborated in these articles. We hope that inclusion of
this topic in a book on forest tissue culture will acquaint new
readers with in vitro pathology and show what research tools are
and may become available.
 209

Tissue culture systems provide a means to study such things

as the infection processes, the involvement of phytotoxins and
phytoalexins in the disease reaction, and to define and segregate
the various tissue and cellular disease resistance mechanisms.
On the more practical side, these systems can permit production
of virus free plants, the maintenance of obligate parasites which
only survive in host tissue and the opportunity for schemes to
select for pathogen virulence or host resistance. The use of
tissue culture systems to gain an understanding of resistance
mechanisms and to develop assays for disease resistance is an
enticing one, especially to foresters who must depend on
resistant planting stock. However, effective advances in this
methodology are confronted by the proverbial problem that
cultured cells and tissues do not necessarily act like the plant.
Resistant cultures often produce plants which fail to be
resistant in the field and vice versa. Understanding and control
of this phenomenon would constitute a major breakthrough.
Ingram (44) pointed out that clear demonstraton of the
utility of resistance expression in vitro awaited study in
systems where host and pathogen background information was
extensive. Helgeson and Haberlach (32) have recently reiterated
this point in discussing the general features of ideal systems
for in vitro resistance work. They recognized environmental
regulations, media composition and inoculation procedures as
important, but first and perhaps most importantly they recognized
the advantage of studying a system with well . defined host and
pathogen genetics. Forest pathologists do not have a wealth of
information on either the genetics or physiology of forest trees
and pathogens, but wise use of the many other in vitro approaches
should speed up the gathering of this information. The
horticultural and agricultural industries have already benefited
from tissue culture pathology. Hopefully, forest pathologists
will likewise seize the opportunity to apply these methods for
applied and basic research.
210

2. PATHOGEN CLASSIFICATIONS
2.1. Viruses
Perhaps no other area of plant pathology has found tissue
culture more useful than has virology. This area has benefited
greatly from tissue culture in both applied and basic research
areas. Applied benefits have been realized through virus
elimination research, which has the production of virus free
(disease-free) plants as its goal. Basic resesarchers initially
viewed tissue culture as promising for a convenient and
environmentally regulated means of obtaining the host cells
necessary to sustain viruses. However, conventional callus
cultures proved difficult because infection establishment was
inconsistent, and even when infection was obtained, the titre
frequently decreased (43). Infection of callus generally
depended on abrading the callus surface and inoculations were
unquantified (46). The use of plant protoplasts (cells devoid of
cell walls) currently is proving workable for virus studies, and
providing the benefits of convenience and regulation originally
perceived by researchers. Due to the absence of the cell wall on
protoplasts, plant virologists are now able to get synchronous,
efficient in vitro infections which are amenable to quantitative
study (46).
Protoplast techniques are not yet available for all plant
species, and in some cases obtaining and maintaining protoplasts
is difficult. However, despite tissue culture difficulties the
use of protoplasts for viral infections has become widespread,
and several reviews (44, 45, 89, 90, 100, 102) are available on
protoplast-virus interactions.
The efficiency of protoplast infection relative to infection
of cells with intact cell walls is currently fostering study into
several areas: (a) Initial infection studies with protoplasts
which have examined viral uptake as a function of pinocytosis
(17) or plasmalemma injury (11, 12), (b) Double infection
studies,where protoplasts were simultaneously inoculated with 2
viruses (76) or where systemically infected protoplasts were
inoculated with a second virus (4, 5). Works such as these
should produce better understanding of cross protection. (c)
 211

Virus replication studies (100). Synchrony of infection has

greatly aided recognition of sequential aspects of replication.
(d) Disease resistance studies showing that virus-protoplast
systems are useful. Hypersensitivity was noted in tobacco
protoplasts challenged with tobacco mosaic virus (75), and Wood
et ale (101) are developing procedures for cucumber and cucumber
mosaic virus which will allow direct comparison of infection and
replication events in both susceptible and resistant lines.
Virus elimination, and the production of disease free plants
through tissue culture has become common practice in the
horticultural industry as a way to avoid yield and vigor
reductions caused by diseases. A variety of methods, each of
which may be partially or totally successful at virus
elimination, including callus culture, protoplast culture,
meristem tip culture and others (94) can be used for producing
disease free plants, and the lists of plants freed of viruses via
tissue culture are impressive (77, 94, 95). Although many
approaches are available for virus elimination, meristem tip
culture (sometimes coupled with thermotherapy or chemotherapy) is
the method most used due to the genetic stability and consistent
clone phenotype of tip culture methods (94). A good review of
meristem tip culture methods (71) and a diagrammatic scheme
generally applicable to all plants detailing elimination
procedures (95) are available.
Meristem tip culture, like any tissue culture method which
utilizes preformed buds, generally produces uniform plants (66,
67). This is of course necessary if the objective is to produce
large numbers of identical plants. Conversely, regeneration of
plants from callus cultures does not provide as much uniformity
(66, 67, 71) as previously noted for tip culture. When callus
cultures are used to regenerate plants as in virus elimination,
variant plantlets may arise from a single parent clone (31, 58,
74)
Berbee et ale (6) produced plantlets via tissue culture in a
forest species, poplar. Subsequently, Berbee et ale (7)
regenerated virus free (symptomless) plants from an infected
parent via callus cultures. They noted high variability in
212

growth rates and branching habits within a sub-clone (one of

multiple clones initiated from the same parent) and even among
plants derived from a single callus culture. They speculated
that such variation may be due to complete vs. incomplete
pathogen elimination or more likely have a genetic basis.
Thus it would appear that virus elimination methods may
allow a unique opportunity to use variability or uniformity in
regenerated plants. One may select the constancy of meristem tip
culture when needed, or one could search for useful variations
from callus culture eliminations. Both cases can provide the
added benefit of disease free stock. Virus elimination has
already been demonstrated for poplar (7), and theoretically is
available for any tree which can be regenerated by tissue
culture. We do not yet know of any studies dealing with tree
protoplast-virus interactions, but this may be changing quickly,
as methods for producing protoplasts from forest trees are
starting to emerge.

2.2. Bacteria
Studies dealing with bacteria in tissue culture systems are
not so numerous as those reported for fungal and viral
associations, but for several bacterial pathogens, studies on
disease establishment and host resistance are receiving
attention. Persistent effort has been applied since the 1950's
(13) to in vitro study of the tumorigenic bacterium Agrobacterium
tumefaciens, but recently the attention has shifted to the
molecular study of the DNA plasmid vector of the disease.
Studies on association of nitrogen fixing bacteria with plant
cells (18) and symbiotic relationships with tissues and organs
(91) have recently been reviewed. This work is also shifting
substantially toward the genes involved, but the fundamental
aspects of symbiosis remain unclear. Tumorigenesis and nitrogen
fixation are special cases for special purposes, but they helped
to form the methodology for studying bacteria in tissue culture
systems .
Infection and toxin induced disease symptoms were
demonstrated in vitro in 1953 with soft rot bacteria and callus
-- -----
 213

from several species (93). Recent studies (38) are of interest

with regards to bacterial toxins. Toxin (amylovorin) production
was demonstrated in apple suspension cultures infected with
virulent strains, but not with avirulent strains of Erwinia
amylovora. The toxin thus produced caused wilting of susceptible
host apple shoots but no wilting in non host tobacco and tomato
shoots. Hsu and Goodman (39) have also used apple suspension
cultures to study toxin related agglutinating factors produced by
virulent strains of ~. amylovora. In these studies, the cultured
cells interacted with the bacteria to produce toxins typical of
the plant diseases. The processes involved in these interactions
are more open to study in controlled culture than in the tree.
Huang and Van Dyke (40), using the scanning electron
microscope, studied tobacco callus responses to inoculation with
three species of Pseudomonas. Pseudomonas tabaci, a tobacco
pathogen, rapidly colonized tobacco callus whereas P. pisi
(pathogenic on pea but not tobacco) multiplied slowly and was
trapped in a network of fibrils on the callus. Non-pathogenic
(saprophytic) ~. fluorescens was not trapped in fibrils, but grew
slowly and was deformed, perhaps a consequence of bacterial cell
wall damage (40). This study demonstrated differential host
response at the cellular level to various inocula. Similarly,
Ruyak et al. (81) demonstrated resistance and susceptibility to
two races of Xanthomonas malvacearum by cotton callus and
suspension cultures from known resistant and susceptible lines
respectively.
Studies dealing with crown gall, Agrobacterium tumefaciens,
are abundant. Butcher (13) reviewed many aspects of the
chemistry, physiology and tumor establishment in A. tumefaciens.
Butcher (14) updated that review, citing research relating the
bacterial plasmids contained in A. tumefaciens to the expression
of tumorigenesis. Schilperoort et al (85) suggested that Ti
plasmids alone were capable of tumor induction, and now the role
of Ti plasmids is widely accepted in the establishment of crown
gall tumors. Most recent reviews by Butcher et al. (15) and
Davey et al. (19) provide good literature reviews and methodology
for working with A. tumefaciens in tissue culture. Although
214

crown gall disease can occur on some tree species (see Butcher,
14), it is mentioned in this chapter mainly due to its importance
as an example of a plasmid capable of transfer of foreign DNA to
a higher plant (19). The Ti plasmid may be viewed as a model
system to act as a guide for those who seek genetic engineering
of forest trees.

2.3. Nematodes
Dual or monoxenic culture of plant parasitic nematodes and
host tissues had its foundation in the early 1900's, but Mountain
(69) in 1955 provided impetus for further work by growing
Pratylenchus minyus in excised root cultures of tobacco and corn.
Since that work, reports of nematodes in cultures of excised
roots have been many, and the culture of certain nematodes on
callus tissues is routine (21, 59, 86). In general, migratory
nematodes can be routinely maintained in callus cultures, but
sedentary endoparasitic nematodes can not yet be fully maintained
on callus alone (53). Interest in the in vitro dual culture of
nematodes grew through the 1960's and early 1970's and
information was gained about parasitic nematodes and their
disease interaction with host cells, but attempts to refine
cultural methods to the axenic level produced little success. As
a result, few plant parasitic nematodes as yet can be maintained
in axenic culture free of host tissues (70, 92). In contrast to
the parasitic nematodes, free living nematodes are more amenable
to axenic culture and methodology for the culture of the free
living species has been reviewed (73, 80). The information
gained from studies with free living forms will provide base
information to future axenic studies with plant parasitic forms.
Currently, dual cultures of plant tissues and nematodes are
important in producing large stock quantities of nematodes for
experiments, or in maintaining "nematode banks" of defined
nematode races or populations (8). Dual cultures also have been
useful in the study of host-parasite interactions, especially
with regards to plant growth regulators which apparently affect
both the nematode and the host. Schroeder and Jenkins (86)
demonstrated better reproduction of Pratylenchus penetrans in
 215

callus cultures than in excised roots. Webster and Lowe (98)

suggested that 2,4-D increased tissue susceptibility thus
indirectly aiding reproduction. Conversely, Krusberg and
Blickenstaff (60) reported that kinetin directly enhanced
Ditylenchus dipsaci reproduction. Hormonal activities in
nematode infected plant tissues are perhaps better understood
today as a result of the in vitro work. Early work by Sandstedt
and Schuster (83) with carrot discs infected with root knot
nematodes questioned polar transport of auxin in infected
tissues, and later (84) they suggested from studies with tobacco
callus that these nematodes did not secrete auxin. Instead it
appeared likely that the transport of auxin past infected areas
was hampered, thus causing accumulation and callus production at
infection sites. They noted, however, that regulation of auxin
in infected tissues involved more than just inhibited transport.
Recently, Jones (54) demonstrated that auxin protectors
(alternative substrates for IAA oxidase) stimulated by nematode
infection in callus may effectively serve to increase auxin
levels. Findings such as these on growth regulator transport and
protection may go a long way towards explaining the galls and
swellings associated with some nematode infections. ~ vitro
studies also have been used to research disease symptomology,
disease resistance, and nematode behavior [see Zuckerman (103)
for an excellent review of cultural studies with nematodes).

2.4. Insects
Judging from the paucity of literature in the area, it
appears that insect-plant dual cultures are little used in plant
pathology. Ingram (43) in reviewing plant parasites in tissue
culture cited only 5 papers which dealt with both insects and
plant tissues in vitro. In later reviews Ingram (44, 45) either
eliminated consideration of insect-plant tissue culture work or
maintained without expansion his previous consideration.
Recently, however, Mott et al. (68) and Nappen (72) have used
loblolly pine tissue culture to study interactions with beetles.
Mott et al. (68) demonstrated that southern pine beetles
Dendroctonus frontalis could be reared from eggs in vitro on a
216

diet of loblolly pine callus alone, thus questioning the role of

mycangial fungi with regards to nutrition. Nappen (72) attempted
to improve the methods of Mott et ale and extended them to Ips
bark beetles; however, only 6% survival was obtained with Ips
beetle as opposed to 26% survival (68) with southern pine beetle.
Thus meth9dology for studying various pine beetles in vitro is
promising, but still risky. The difficulties seem to center on
providing adequate physiological and behavioral environments for
the insect which are also adequate to host culture survival.
2.5. Fungi
2.5.1. Dual and axenic culture studies. A major limitation of
working with plant pathogenic fungi has long been the complexity
of working with intact infected plants. It would be simpler to
work with only the needed parts of the plant and in carefully
controlled culture environments. Comlexity would be further
reduced if the fungus were axenically cultured to provide a
constant source of uniform, contaminant-free inoculum. Both
avenues can be utilized in tissue culture pathology but obstacles
of methodology and interpretation presently limit free
application of this approach. However, the promising rewards of
being able to study cloned axenic fungi inoculated on cloned
cultured host tissue in controlled environments continues to
spark active interest.
Axenic cultures can be obtained easily for fungi which grow
readily as saprobes. However, the traditional obligate
parasites, such as Uredinales, Peronosporaceae and Erysiphales,
which are not readily capable of saprophytic growth, have
presented problems that required much effort in the areas of dual
and axenic culture (see Ingram (45) and Scott (87) for good but
not comprehensive lists of dual and axenic cultures,
respectively). Dual culture of fungus and host together was
first successful in 1944 for culture of Plasmopara viticola on
grape, Vitis vinifera (65). In subsequent years tens of fungal
species were dual cultured but specific rules for initiating
cultures are still difficult to establish.
 217

Dual cultures may be established from pieces of infected

host tissue or by the in vitro inoculation of cultured host
tissues with one of a variety of contaminant-free fungal
materials such as spores, hyphae, or sporangia. Obtaining
contaminant-free inoculation materials is a problem, but one that
can usually be overcome. Inocula from axenic cultures, of
course, present no problem.
Direct in vitro infections are common with intact host
tissues and organs, but callus and tissues without epidermis have
proven variable. Ingram (43) presents a good discussion of cases
where direct infection was limited by the absence of epidermis or
cuticle. This illustrates the general point that all aspects of
the fungus-host interaction may not be available for study in
vitro, depending on the nature of the host culture. It also
suggests that with suitable selection of the type of host
culture, specific parts of the total intact plant response may be
unraveled and studied separately.
Illustration of another general point is at hand. In vitro
responses must be interpreted with care, for lack of infection in
the absence of epidermis does not necessarily imply a causative
relationship. Antimetabolites may be equally, or perhaps more,
important in controlling in vitro infection than the presence or
absence of an epidermis. Maheshwari et al. (61) were unable to
infect snapdragon callus with Puccinia antirrhini. Although
absence of surface features may have been important, failure of
uredospore inoculum to germinate on the callus or on nutrient
agar near the callus strongly suggests some inhibitor from the
callus since uredospores germinated in 3 hr on water agar. Many
researchers have noted antimicrobial agents in cultured tissues
or in the media (16, 50, 51, 52, 56, 63, 82).
Examination of the in vitro interactions of loblolly pine
and Cronartium quercuum f. sp. fusiforme, causal agent of
fusiform rust disease, provides a good demonstration for fungal
inhibitors. Loblolly pine embryos (ca. 14 day old seedlings) can
be routinely infected in vitro with basidiospores via methods
elaborated by Amerson and Mott (2). These methods have been used
to infect embryos from both resistant and susceptible seed
218

families (3) at the 95% level (Amerson and Mott, unpublished

data), and Jacobi et al. (52) demonstrated embryo infection on 5
different media. Thus, intact embryos are highly susceptible to
in vitro infection. In contrast, loblolly pine callus is immune
to surface colonization and/or haustorial penetration. Jacobi et
al. (52) were unable to obtain colonization or infection of
loblolly callus on any of 7 med i a tested, and during four years
of research with this system, in vitro infection of callus has
never been observed (Amerson and Mott, personal observation) .
One might speculate that the lack of infection was due to the
absence of epidermis or cuticle in callus, but Amerson and Mott
(unpublished data) have demonstrated infection of cortical
parenchyma in embryos which were longitudinally split to expose
interior tissues, and in embryos stripped of overlying epidermis.
Admittedly, the surface of differentiated parenchyma differs from
callus, but this direct parenchymal infection does cast doubt on
the necessity of epidermis for ~. quercuum f. sp. fusiforme
infection. Likewise, early studies (96) showed that, although
this rust may survive in systemically infected slash pine stems
in culture, it would not spread to callus initiated on these
stems. Maheshwari et al. (61) made similar observations with
sunflower explants systemically infected with a rust. Again the
importance of epidermis appears doubtful.
Investigations of loblolly pine callus immunity (50, 51, 52)
support the contention that toxic metabolites are produced by and
liberated from callus. Jacobi and co-workers demonstrated that
established axenic hyphal colonies of C. quercuum f. sp.
fusiforme failed to grow on loblolly pine callus even though the
support medium was suitable for both organisms. Furthermore,
colonies placed on the medium near callus or callused embryos
were inhibited whereas colonies near uncallused embryos were not.
The inhibitory factor(s) was extractable in aqueous solution from
loblolly callus, and embryos grown in the presence of callus or
extracted inhibitors exhibited a lesser degree of infection than
controls. Attempts to characterize the inhibitor(s) are
incomplete but the evidence clearly points to some fungal
inhibitor as limiting infection of cultured callus cells.
 219

Consideration of research to establish axenic cultures of

"obligately" parasitic fungi may seem beyond the purpose of this
chapter; however, the establishment of axenic cultures is a
logical extension of dual culture work and a point where forest
pathology has been active. Apart from the rusts, Scott (87)
lists only one downy mildew as having been cultured from groups
traditionally considered as obligate parasites (Peronosporaceae,
Albuginaceae and Erysiphales), and Alexopoulos and Mims (1) list
only two downy mildews. Thus this section will dwell only on the
rust fungi, Uredinales.
Hotson and Cutter (37) obtained the first axenic rust
cultures by establishing the rust Gymnosporangium juniperi-
virginianae from infected host tissues. Williams et al. (99)
were the first to obtain rust cultures (Puccinia graminis f. sp.
tritici) directly from spores. Today, although not routine,
several rust species from at least 5 genera can be cultured.
Studies of these rusts, mostly cereal and flax rust, have
provided information on nutritional requirements, inoculum
density and colony formation, the importance of infection
structures to colony establishment, and other factors (87) which
serve to increase although not guarantee the chances of culturin~
a particular rust. Of particular interest in this chapter are
the rusts Cronartium ribicola (white pine blister rust) and
Cronartium quercuum f. sp. fusiforme (fusiform rust) which occur
as pathogens of various five and three needle pines,
respectively. Both are devastating rust diseases which have
caused many millions of dollars of damage in pine forests. Both
rusts can be grown in axenic culture. Harvey and Grasham (27,
28) established dual cultures of systemically infected pine stems
and of spore inoculated callus, respectively, and in 1974 they
obtained mononucleate axenic hyphal cultures from infected stem
callus (30). Recently, Diner et al. (20) reported techniques for
obtaining axenic mononucleate hyphal cultures directly from ~.

ribicola basidiospores at nearly 100% efficiency. Axenic

colonies of ~. quercuum f. sp. fusiforme have been established
from basidiospores (2, 25), uredospores (25, 50), and aeciospores
(25). In one study, Hollis et al. (36), on very rare occasion,
220

obtained axenic hyphal colonies from infected slash pine stem

segments, but this work has not been repeated. The growth and
study of these two pathogens in dual and axenic culture has
proved interesting, but more importantly in vitro techniques are
being applied with the intention of using tissue culture as a
vehicle to tree improvement. As discussed in a subsequent
section, axenic cultures of ~. ribicola are already being used
for disease resistance research and other uses will surely arise.

2 . 5.2 Disease resistance studies with fungi. The introduction

to this chapter noted, as have previous works (32, 44), that
detailed information about the host and pathogen are extremely
helpful in understanding and interpreting disease resistance
studies. Indeed all host materials that enter culture as disease
resistant or susceptible do not necessarily test-out resistant or
susceptible in properly corresponding fashion in vitro (34).
This is not uncommon, several studies on susceptible or resistant
lines tested contradictory in culture (42, 49). In contrast,
several host-pathogen systems have displayed fidelity of disease
expression in culture (33, 41, 48, 97).
Special attention should be paid to two studies Helgeson et
al. (33), Helgeson et al. (34). Helgeson et al. (34), working
with Phytophthora parasitica var. nicotianae race 0 and callus
cultures of tobacco from resistant and susceptible varieties,
demonstrated under prescribed conditions that resistance was
faithfully expressed. Under these prescribed conditions (tight
callus morphology, inoculum 3-30 spores/ml, and temperature
generally 20-24C) callus from plants known to be resistant to
race 0 consistantly showed slower colonization than callus from
susceptible plants when challenged with race O. However, when
challenged with race 1, a race pathogenic to both tobacco
varieties, equal colonization of the callus was obtained.
Elevation of incubation temperatures to ca. 27 or 28C, changes
in growth regulators, changes in callus morphology and increased
inoculum density all served to reduce or eliminate the
differential colonization of race 0, and thus resistance
expression. This paper vividly demonstrated the pliable nature
 221

of resistance expression in vitro, and its reliance upon culture

conditions. At the same time it points out that faithful
expression can be obtained with defined culture parameters.
Despite this demonstration of fidelity, proof that the in vitro
resistance was due to resistance genes normally expressed in the
intact plants was not obtained until 1976 (33). In this study,
plants homozygous resistant and homozygous susceptible to race 0
were used to produce selfs, Fl' F2 and F3 plants, thus
allowing for segregation. In all cases, resistance tests with
cuttings and callus from 185 plants produced the expected
results. Resistant plants produced only resistant callus and
susceptible plants produced only susceptible callus when
challenged with race O. Challenge by race 1 produced
susceptibility in all plants and callus tested, as expected.
Thus it seems clear from this work that genetic resistance was
expressed in culture. Alteration of this resistance expression
in the 1972 study (34) serves to point out the opportunity for
error in tissue culture studies of resistance. This opportunity
for error should breed caution, not avoidance.
Having spent several paragraphs stressing the importance of
knowing your systems in the study of resistance fidelity, we
would like now to diverge from that viewpoint and talk briefly
about disease resistance selection where detailed background
information is absent. In selection programs one typically seeks
to recover from culture some new resistant variety. This new
selection depends on changed physiology, genetics, or both, and
this dynamic system mitigates prior knowledge. Resistance
selections of this type, dependent on in vitro generated tissue
changes have been reported for several host plant-fungal systems.
Gengenbach and Green (22) and Brettell et al. (10) working with
Texas male sterile corn and Helminthosporium maydis race T have
selected toxin resistant callus cultures from originally
susceptible tissues. In a subsequent study Gengenbach et al.
(23) again selected toxin resistant callus, and regenerated
resistant plants from this resistant callus. Thus a system where
disease resistant plants were generated from originally
susceptible tissues, screened in vitro, does exist.
222

Unfortunately the gain of disease resistance was accompanied by

restored male fertility (23); thus these plants have little
practical value.
More commonly, tissue culture has been used to generate
variant tissue lines from disease susceptible plants, followed by
in vivo screening of regenerated plants. Heinz et al. (31) in
reviewing sugar cane tissue culture noted such resistance
selection for eyespot disease (Helminthosporium sacchari) and
downy mildew (Selerospora sacchari) where the parent sugar cane
tissues were susceptible, and some of the regenerants were
resistant. Recently, Matern et al. (62) and Shepard et al. (88)
selected for resistance against Alternaria solani and
Phytophthara infestans, respectively, in plantlets produced from
protoclones (protoplast cultures) of the susceptible potato
variety, Russet Burbank. In all of the selections cited,
knowledge of both the host and pathogen genetics would help in
understanding the basis of observed resistances, but generation
of the resistant selections appears possible in almost any
system.
Disease resistance research utilizing tissue culture of
forest trees has not been extensive but the Cronartium-Pinus (2,
26), Ceratocystis-Ulmus (35) and Castanea-Phytophthora (9)
interactions have received some study. Advances in the culture
of American chestnut (55) provide a basis for work on chestnut
blight. As previously noted, cultured loblolly pine callus is
immune to infection by ~. quercuum f. sp. fusiforme regardless of
the parental resistance, but intact embryonic materials are
generally susceptible. Little published information exists on
these embryo infections, but studies currently in progress (Mott
and Amerson) seek to characterize infection, catalog observed
resistances, and develop in vitro resistance assays. Loblolly
embryos have been used in vitro to demonstrate necrotic reaction
zones (Amerson and Mott, unpublished data) similar to those
observed in greenhouse studies (64). The in vitro pine embryo
system was used by Gray et al. (24) to describe the
ultrastructure of the haustorial apparatus of ~. quercuum f. sp.
fusiforme, and ultrastructural studies in progress (D.J. Gray,
 223

unpublished data) have noted host cell and haustorial necrosis,

and haustorial exclusion via deposition of a callose wall
apposition.
Early research with C. ribicola focused primarily on the
establishment of infection, but more recent studies have a
disease resistance orientation. Harvey and Grasham (27)
demonstrated that western white pine callus was susceptible to in
vitro infection by ~. ribicola. Subsequently, Harvey and Grasham
(29) showed infection of the non-host species Douglas-fir. Robb
et ale (78, 79) perhaps began the resistance work with their
ultrastructural study of host cells changes in the Pinus
monticola - ~. ribicola system. More recently, Harvey (26)
demonstrated apparent pathotoxin production in a ~. monticola -
~. ribicola co-culture in which host and pathogen were physically
separated by a dialysis membrane. Demonstration of the
pathotoxin should facilitate future resistance work in this
system. Diner et ale (20) improved axenic culture techniques for
C. ribicola and used these cultures for resistance studies with
resistant and susceptible (57) sugar pine embryos and callus.
Susceptible embryos challenged with hyphal cultures in vitro
(Diner and Mott, unpublished data) were easily infected, with no
visible host response, whereas resistant embryos displayed
characteristic hypersensitive necrosis and cellular collapse. As
with the embryos, infection in susceptible sugar pine callus was
widespread with no visible host response. Callus from resistant
plants became infected, but hyphal and haustorial spread was
limited to a region of densely staining cells not seen in the
susceptible group (Diner and Mott, unpublished data).

3. CONCLUSION
In a chapter such as this where the topic is very broad and
the purpose is mostly elaboration of research approaches or
tools, it is difficult to decide when to stop adding topics for
consideration. By no means have we covered the overall area of
in vitro pathology. Topics such as parasitic higher plants are
not discussed here nor are well studied areas such as phytoalexin
or mycorrhizal research considered. However, the reader is
224

referred to an excellent book, Ingram and Helgeson (47), for a

review of these last two areas and an introduction into the
literature. Likewise, areas such as nitrogen fixation and crown
gall studies have received only light emphasis, but the scope of
this chapter precludes in depth consideration and the reader is
again referred to reviews cited with the appropriate section.
It is obvious from this brief review that pathological
studies with plant tissue cultures are becoming commonplace with
agricultural and horticultural species. As plantlet
regeneration, protoplast fusions and genetic engineering further
advance, more opportunities to utilize pathological studies will
arise. Forest pathologists should take a careful look at tissue
culture research, as an alternative to whole tree research. The
control and perhaps simplified systems offered by tissue culture
can be applied to improvement areas such as disease resistance
selection as demonstrated in agriculture. This review notes the
fact that few forest pathology studies have been undertaken. The
few cited, Berbee's et al. (7) work with viruses and poplars and
studies with Cronartium have proven useful. Hopefully many more
will be forthcoming.
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 225

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 227

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50. JACOBI WR 1979 Interactions of cultured callus and
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51. JACOBI WR 1981 Inhibition of Cronartium fusiforme by
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52. JACOBI WR, HV AMERSON, RL MOTT 1981 Microscopy of
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53. JONES MGK 1980a The interaction of plant parasitic
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228

56. KHANNA P, A UDDIN, GL SHARMA, SK MANOT, AK RATHORE 1976

Isolation and characterization of sapogenin and solasodine
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57. KINLOCH BB, M COMSTOCK 1980 Cotyledon test for major
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 231

9. ACTION OF GROWTH REGULATORS

J. B. zaerr and M. o. Mapes

1. INTROoocrION

All tissue and organ culture systems used with forest species make
use of natural or artificial plant growth regulators. Without added
hormones, nost tissues do oot remain viable, nuch less grow in the
manner we wish. Unfortunately, few studies have dealt with the
mechanism of hormone action; instead, effective hormones and their
concentration have been derived empirically. we are therefore left
with a wide range of hormones that have been applied to a number of
species with sometimes conflicting results. The purpose of this
chapter is to summarize some of these results, particularly those from
recent papers, and arrive at generalizations where possible. Since
extensive reviews are available elsewhere (9, 13, 89), 00 attempt will
be made here to list every paper which has reported use of a growth
regulator in an aseptic culture system of a forest species.
one of the nost striking features of plant hormones is their
multiplicity of effects. Indole-3-acetic acid (IAA), an endogenous
hormone in higher plants, causes cell elongation, but it also affects
xylem formation (78), germination (94), and a variety of other
physiological processes. Gibberellins also cause cell elongation, but
in addition they affect certain flowering processes and seed
germination. Cytokinins are thought of primarily as expediters of cell
division, but they also exert strong control over norphogenetic
processes, as we shall see later. This nultiplicity of effects is
intriguing, but it complicates attempts at explaining how growth
regulators operate.
Tissue culturists have been concerned primarily with norphogenetic
effects of growth regulators. As we look nore closely at the various
classes of these regulators on the following pages, we should keep in
232

mind that these substances may react differently in intact plants than
in culture systems.

2. AUXINS
2.1 Background
A variety of compounds have been classed as auxins by physiol-
ogists. In this chapter we shall consider auxins to be oompounds which,
like some natural hormones, cause cell enlargement in intact tissues.
This classification is based on the morphological response of tissues
rather than on the mode of action of the chemical. '!hus, the
well-known herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) is
considered to be an auxin even though it is rot endogenous to plants.
In culture systems, though, it often produces results similar to those
produced by indole-3-acetic acid (IAA) and is used po substitute for
1M because it is more stable and sometimes more effective. Other
man-made substitutes for IAA such as naphthaleneacetic acid (NAA) or
indole-3-butyric acid (IBA) are also oommonly used for the same
reasons.
Auxins are often used in aseptic culture systems to induce callus
tissue, although the early work of Gautheret (40) with a variety of
tree species clearly indicates that auxin is rot essential in such
cultures. Of course, the large plant p3.rts used by Gautheret may have
contained sufficient native auxin so that there was ro need for an
exogenous supply. Auxins stimulate callus formation, but the optimal
concentration varies with species (43). Auxin is also used to maintain
callus cultures (13). Q'lce callus is obtained, auxins may be used in
conjunction with cytokinins to induce organogenesis (8, 29). It seems
curious that auxins are regularly called upon to promote
dedifferentiation of cells, and then to promote differentiation of the
dedifferentiated tissue. This dual vole of auxins has puzzled
physiologists for many years, and its matter-of-fact acceptance by
current researchers has rot helped to clarify our understanding of
auxin action.

2.2 Indole-3-acetic acid (1M)

1M was the first substance to be recognized as an auxin and has
since been found to be ubiquitous in higher plants . Physiologists
 233

have, at ooe time or another, linked it to alnost every known

physiological process (94). It is usually associated with cell
enlargement, but its involvement in morphogenetic processes such as
root formation are readily demonstrated nor a variety of species (97).
IAA action has long been associated with IAA oxidase activity, and
recent evidence suggests that peroxidase-mediated control of IAA may be
one of the major mechanisms controlling auxin levels and, thus, auxin
action (102). Since the gibberellins appear to modulate auxin activity
through IAA oxidase levels, such a system would account for the similar
effects of both hormones 00 cell elongation (54).
Other mechanisms of IAA action are also possible. For example, IAA
loosens cell walls within a few minutes after application, presumably
by modification of cell membranes. Little is known about the IAA
receptor sites in membranes, but they do not appear to be analogous to
the proteins acting as receptors for hormones in animals (102).
Binding experiments now in progress in several laboratories may soon
clarify this point. Another mechanism which could control the quantity
of IAA present in a tissue is the formation or dissolution of
conjugates of IAA, such as indoleacetylarabinose, indoleacetylglucose,
IAA-myoinositol, and proteins (102). This mechanism is long overdue
for investigation.
The close relationship between IAA effects and RNA synthesis leads
to the conclusion that IAA somehow activates preformed enzymes. The
extension growth induced by low Pi and the apparent change in Pi
induced by auxin suggest that these treatments may bring the Pi of the
cell closer to the Pi optimum of certain critical but as yet
unidentified enzymes and thus induce growth (102).
IAA is the most interesting of all of the auxins frequently used
in aseptic culture systems because it is the only one found in nature.
Unfortunately, it is easily oxidized and is unstable in light. The
actual concentrations to which cells are exposed are therefore seldom
if ever known. The o::mnon practice of using synthetic auxins such as
2,4-D or NAA as substitutes for IAA may be more effective for certain
purposes such as inducing callus, but such practice also clouds our
understanding of how IAA brings about its manifold effects and in
particular how IAA functions in intact plants. Fbr these reasons the
preferred auxin is IAA when it can be used (72).
234

1M is often used in rredia for ootaining callus fran forest tree

species (13, 43, 56, 61, 66, 109, 110, Ill, 112, and many others). Of
mre interest, however, is the use of IAA to pranote mrphogenesis and
cell differentiation in cultures. IJoo and wang (63) showed that
embryos of Pinus yunnanensis and Kiteleeria davidiana developed boward
maturity \\hen treated with IAA. '!his "native" auxin has since been
reported to induce shoots in cultures of Pseudotsuga menziesii (25),
Tsuga heterophylla (26), Santalum album (74), Betula pendula (47), and
Picea abies (3, 4, 5). Roots were induced in Tectona grandis (42),
Pinus lambertiana (41), and Populus tremuloides (116) ~ both roots and
shoots were produced in cultures of Eucalyptus alba (56), several spe-
cies of Populus (2), Pinus sylvestris (10), Pinus lambertiana (41),
Betula pendula (47), Santalum album (6), and Pseudotsuga menziesii
(11). New xylem was stimulated in Pinus sylvestris (118), tracheids in
Cryptaneria japonica (66), and fOllen tube growth in Pinus roxburghii
(36).
IAA did rot induce roots in Castanea sativa (101), and it
inhibited induction of rods en reedles of Picea abies (5). It also
inhibited formation of male strobili in explants of '!huja plicata (33).
Winton (108) reported that both roots and shoots were produced fran
callus of Populus tremula without the use of any auxin at all. '!hese
resul ts do rot rrean that IAA is rot required for organogenesis or that
it is inhibitory~ the tissue or explant itself could be producing suf-
ficient IAA to cause organogenesis. If such were the case, additional
IAA in the culture rredium could then be ineffective or even inhibitory.
Unfortunately, questions raised by these experiments cannot be
answered without rreasuring the levels of IAA in the rredia and in the
tissues involved before and during the course of morphogenesis. Such
experirrents are rot easy to conduct and, until recently, suitable
techniques for the rreasurements of IAA were rot available. Recent
improvements in analytical techniques employing rapid and efficient
purification procedures and sensitive fluorescence detectors now make
possible definitive rreasurements of IAA in sub-gram quantities of
tissue (96). Further developments in the use of radioimmunoassay
techniques will also expedite rreasurerrents of IAA (104). 'lbese
techniques may result in a new generation of experiments to clarify the
role of IAA in organogenesis.
 235

2.3 Indole-3-butyric acid (lEA)

The nost cx:mnon use of IBA in aseptic culture systems has l:een as
a substitute for IAA. IBA is quite sbnilar to IAA in nolecular
structure, differing only in the length of the side chain. It is nore
stable than IAA, and it is equally or nore effective as an auxin for
certain purposes than is IAA. Its resistance to oxidation may account
for it superior effectiveness in certain culture systems. It is
particularly associated with vooting. Winton and Verhagen (114), for
example, used IBA to induce vooting on shoots derived fran callus of
Pseudotsuga menziesii, and Sita (85) used it to induce voots on shoots
derived fran callus of Eucalyptus citriodora. It has also l:een found
to induce shoots in Pinus strobus (68) and Pinus gerardiana (58) and
callus, voots, and shoots in Pinus sylvestris (10). 'Ihese are rut a
few examples of the wide use IBA has found in aseptic culture systems
for fiJrest trees.
Although lEA appears to substitute for IAA satisfactorily, its
effects may in fact l:e slightly different fran those of IAA. von
Arnold and Eriksson (3), for example, shCMed that in Picea abies, IAA
produced a light-green callus while IBA produced brown callus. These
results suggest that the two compounds are not strictly
interchangeable; the difference may l:e due to the higher rate of
destruction of IAA by oxidative enzymes, resulting in different
concentrations of the two hormones in the tissues.

2.4 Naphthaleneacetic acid (NAA)

NAA is another analog of IAA widely used in culture systems.
While IBA has the same ring structure but a slightly longer side chain
than IAA, NAA has a nodified ring structure rut the same side chain as
IAA. Thus, the structure of IBA and NAA roth differ slightly fran IAA
but in different ways. The node of action of NAA is probably similar
to that of IAA, but the fonner is much nore stable than the latter and
for that reason has l:een used extensively as a substitute for IAA.
NAA has l:een widely used to induce callus in roth gymnosperms (90)
and angiosperms (18, 93). It is often the l:est-suited substance
available for this purpose, perhaps l:ecause of its resistance to
oxidation. Roots are cx:mnonly induced on calli as well as on embryos
236

or explants l: NAA {14, 17, 18, 22, 23, 30, 32, 37, 38, 74, 75, 82, 85,
100, 103, 105, 116}.
In some cases, lx::Mever, removal of NAA fran the culture !Tedium
resulted in induction of roots {29}. NAA has been found to aid in
praroting shoots in a few species such as Pinus radiata {75}, Picea
abies {52}, Thuja plicata {33}, Pseudotsuga menziesii {24, 29}, and
Santalum album {6}, rut it also inhibits shoot formation in sane cases
{IS, 17, 37, 116}. Cheng and Voqui {29} found that NAA inhibited root
formation on cotyledon explants of Pseudotsuga menziesii. Konar {58}
reported stimulation of suspension cultures of Pinus gerardiana, and
David and David {35} found an increased yield of protoplasts \'Alen
cotyledon segments of Pinus pinaster were pretreated with NAA. Similar
results were reported with protoplasts of Pseudotsuga menziesii {55}.
NAA is rot a naturally occurring plant honnone, rut it has found
extensive use in the hands of tissue culturists. Even though we do rot
understand its mode of action, without this compound many of the recent
advances in the technology of tissue culture probably would rot have
been forthcaning.

2.5 2, 4-dichlorophenoxyacetic acid {2,4-0}

About 150 million pounds of 2,4-D are used annually in the world
for the ];Urpose of killing unwanted plants {M. Newton, personal
communication}. This potent herbicide has also found extensive use in
aseptic culture systems, \'Alere it often induces cell proliferation.
Perhaps its most rotable use (other than as a herbicide) is to cause
dedifferentiation and unorganized cell growth or callus. Table 1 lists
sane forest tree species on \'Alich 2,4-0 has been used to induce callus.
It is clear that this compound is effective over a broad range of
angiosperms as well as gymnosperms.
Reports of 2,4-0 inducing organogensis are few. Cheng {27} found
shoot induction on cotyledon explants of Pseudotsuga menziesii l:
2,4-0, and Kitahara and Caldas {56} found that it enhances root growth
in Eucalyptus alba and ~. grand is cultures. Venverloo {100} found that
low concentrations {0.01 to 0.05 rrg/l} of 2,4-0 stimulated roth root
and shoot growth in Populus nigra callus cultures rut that it generally
suppressed meristematization processes. Winton (107) reported
stimulation of rooting in callus of Populus tremuloides l: 2,4-0, and
 237

Table 1. Some forest species funning callus upon treatment with 2,4-0.
Angiosperms Gyrmosperms
Citation Citation

Acacia Larex occidental is 43

- - - -koa
- 87
Acer pseudoplatanus 115 Picea abies 3
- ---
Artocarpus heterophyllus 72 P. excelsia 46
Betula pendula 47 ~. glauca 21
castanea sativa 101 Pinus albicaulis 43
Eucalyptus alba 56 P. banksiana 23
E. bancroftii 61 P. cembra var. sibirica 80
E. camaldulensis 92 P. <;,!erardiana 57
E. grandis 56 P. flexilis 43
Populus nigra var. italica 100 P. Einaster 35
P. simonii x P. nigra 2 P. strobus 68
P. tremuloides 106 pseudotsuga menziesii 43
P. ussuriensis 2
Santalum album 74
Tectona grandis 72

aFor additional forest species, see (13) .

Huhtinen and zeki (47) found similar results with Betula pendula.
Mapes (unpublished results) found that a high concentration (2.5 mg/l)
of 2,4-0 caused root primordia to fonn in profusion on hypocotyl
segments of Tsu<;,!a heteroEhylla and that rooting of adventitious
shoots could be cbtained by using 2,4-0. On the other hand, Vieitez et
al. (101) found no effect of 2,4-0 on differentiation in castanea sativa
cultures, and WOlter (116) noted that it inhibited root formation in
Pq>ulus tremuloides callus. Konar (58) reported that 2,4-0 caused
cells in suspension cultures of Pinus <;,!erardiana to proliferate but
that it inhibited root formation on oolid nedia. Chalupa and rurzan
(20) found only inhibition of shoot growth with 2,4-0 in cultures of
Picea <;,!lauca.
'!his brief surrrnary leads to a rather cbvious conclusion: 2,4-0
does not affect plant cells like other auxins, even though it is
238

generally classed as an auxin by physiologists. Its mode of action is

poorly understood and it is, for most uses, not a substitute for
natural auxins. 2,4-0 is effective in inducing and proliferating
callus in many species. Its role as a dedifferentiating substance is
clearly established; thus, it should not be surprising that it is not
particularly useful for inducing organogenesis in most forest species.
Since 2,4-0 has been shown to cause chromosomal changes (84), it should
be used cautiously.

2.6 Other auxins

Several other compounds possessing auxin-like properties have been
errployed in tissue culture systems. Indolepropionic acid (IPA), an
analog of the natural hotlllOne JAA, was found to stimulate root
initiation on callus of Pinus gerardiana (58) and Tectona grand is (42),
but Harvey et al. (44) found that it did not modify the grCMth of Pinus
IOOI1ticola tissue. Risser and White (77) found IPA to be less effective
than either 2,4-0 or NAA in stimulating tumor growth of Picea glauca.
'!he compound naphthoxyacetic acid (IDA) was reported to induce
callus in sections of Eucalyptus bancroftii (61), and it was found to
promote shoot grCMth on callus of Pseudotsuga menziesii (114). It also
stimulated shoot grONth in Santalum album explants (74) rut inhibited
grONth of excised ruds of Picea glauca (21).
Wolter (116) reported that 2,3,6-trichlorobenzoic acid stimulated
root formation on callus of Populus tremuloides, and Rae and Bapat (74)
found enhancement of callus grCMth on Santalum album explants.
Al-Talib and 'lbrrey (1) observed stimulation of shoot grCMth in excised
buds of Pseudotsuga taxifolia (nON R.. menziesii) by
2,6-dichlorophenoxyacetic acid and 2,3,5-trichlorophenoxyacetic acid.
Narasimhan et al. (72) found that 2-benzothiozole oxyacetic acid
stimulated callus development in Artocarpus heterophyllus.
p-Chlorophenoxyacetic acid, an analog of 2,4-0, stimulated callus of
Pseudotsuga menziesii (25).
The variety of "miscellaneous" auxins that appear to elicit
widely differing responses in plant cultures does not help us
understand heM auxins work. Since none of these compounds is
endogenous to plants, \..e cannot be certain that the effects of these
auxins are not akin to distortions caused by plant toxins. 2,4-0 is
 239

certainly a well-established herbicide and is lethal at low

concentrations to a wide variety of species. Naphthoxyacetic acid
prcm:>tes shoot growth in one system (114) but inhibits it in another
(21). Until such ananalies can be explained, we cannot understand how
auxins--natural or artificial--bring about the effects we so casually
use in our aseptic culture systems.

3. CY'IOKININS
3.1 Background
Even before cytokinins were known, it was recognized that a growth
factor other than auxin was required for anbryo development. Coconut
milk was often used to supply that unknown growth factor (73). After
its discovery, kinetin (67) or similar substances soon replaced coconut
milk in many culture systems. The advantages of using defined media
were soon evidenced by rapid progress in aseptic culture technology.
The "hormone balance" ooncept of Skoog and Miller (88), who showed that
the ratio between cytokinin and auxin in the medium was critical in
determining whether shoots or roots were initiated on tobacco callus,
has emerged as a guiding principle for tissue culturists. It is now
recognized that cytokinins are an essential oamponent for culture of
most species and that an appropriate ratio of cytokinins to auxins
often leads to organogenesis.
Cytokinins are closely associated with processes of cell division.
At one time it was thought that cytokinins nodulated tRNA functioning,
but that explanation does rot agree with recent evidence. A IlOre
likely explanation is that cytokinins act directly upon enzymes and
control enzyme activity rather than enzyme synthesis (102). Varnell
and Vasil (99) suggest that kinetin may act on nucleus-based events or
on rrembranes in the cytoplasm in the apical neristem. Whatever the
site of action, recognition of the close tie between cytokinins and
enzyme activity and of the powerful effects of cytokinins on cell
differentiation and IlOrphogenesis has resulted in this group of plant
hormones becoming better understood than any other. The expression of
cytokinin activity is influenced by structural variations in the cyto-
kinins. Spatial oonfiguration as well as the type of atoms on the
N6 substituent determine cytokinin activity. Recognition of this fact
does rot nean, however, that the effects of cytokinins on plant tissues
240

can be pr-edicted. '!he ooly reliable rrethod for learning row any given
tissue is likely to respond to any given cytokinin is by rreans of an
empirical experiment. Recent studies with crown gall disease 00

tobacco and the rapid pr-ogress in genetic engineering techniques pr-o-

mise to elucidate the J1Dde of action of cytokinins (117). These ajvan-
ces should help us understand row to manipulate forest tree cultures
more effectively.

3.2 Kinetin
'!he first cytokinin discovered was 6-furfurylaminopurine, or
kinetin (67). It has been a a::rnponent of many rredia recipes used with
a wide range of forest tree species during the past two decades (14),
even though it is not a natural cytokinin. It stimulates growth of
callus tissue in gymnosperms (12, 43, 80) and angiosperms (51, 61).
In cell suspension cultures of PSeudotsuga menziesii, Winton (113)
reported that kinetin caused a d::>ubling of cell volume weekly. On the
other hand, Chalupa et al. (23) found that kinetin did not improve
callus formation 00 explants of Pinus banksiana, and Risser and \'bite
(77) reported that kinetin inhibited growth of tumors of Picea glauca.
Zajaczkowski (118) found kinetin to have little effect 00 growth of new
xylem in Pinus sylvestris. Numerous "negative" results with other
species have undoubtedly been left unreported.
Because of its IlOrphogenetic effects in tobacco callus (88), kine-
tin has been a logical candidate for use in shoot induction experiments
with forest trees. Either low levels of kinetin alone or kinetin plus
auxin have induced roots in cultures of Castanea sativa (101), Biota
orientalis (95), and POpulus ussuriensis (2). A IlOre OOITIllOn organoge-
netic effect of kinetin, OOwever, has been induction of shoots. Shoots
or buds have been induced by kinetin in cultures of Picea abies (4),
Picea sitchensis (103), Pinus contorta (103), Pinus taeda (65),
Pseudotsuga menziesii (27), Betula pendula (47), Eucalyptus ficifolia
(7), POpulus nigra var. italica (100), ~. nigra var. robusta (19), ~.

tremolo ides (64), ~. ussuriensis (2), and Santalum album (74). In

addition to these IlOrphogenetic effects, Chalupa and Durzan (21)
reported that kinetin encouraged expansion of needles in excised buds
of Picea glauca. en the other hand, Coleman and '!horpe (33) found that
 241

kinetin inhibited the Ormation of male strobili induced by gibberellin

in cultures of Thuja plicata.
This wide range of effects across a broad spectrum of species
makes it difficult to predict exactly what effect kinetin will have on
any given tissue. It rrost ocmronly induces shoots rather than toOts,
but this effect does not always hold. Kinetin is a potent horrrone
that can be usefully employed in certain aseptic culture systems.

3.3 6-benzylaminopurine (SAP)

SAP is related to kinetin in structure, rut it is nuch rrore effec-
tive than kinetin. It has been used extensively in recent years, and
seems to be the favorite cytokinin of tissue culturists. It is stable,
inexpensive, readily available, and--rrost ~rtant--it is highly
effective. Numerous researchers have reported its use in callus for-
mation, rut its rrost ~rtant role is its use in praooting shoot Or-
mation. I t is probably the rrost potent growth regulator available for
this PJrpose. Table 2 lists some of the forest tree species in which
shoots have been induced by B.l\P in aseptic culture. B.l\P has also
induced toOts in castanea sativa (101), Eucalyptus ficifolia (39), Picea
glauca (16), Pinus radiata (75), ~. sylvestris (10), Populus nigra var.
robusta (18), ~. tremula (108), P. tremuloides (107), and Quercus rubra
(82)
It should be noted that in many cases toOt formation Ollowed
shoot Ormation and that the concentration of B.l\P was reduced substan-
tially (lD-fold or rrore) or eliminated c:nce shoots were formed (19, 29,
37, 106). It is ~rtant to keep in mind that even though B.l\P may be
eliminated fran the rredium, carryover of B.l\P may be sufficient to rreet
the needs of the tissue Or a cytokinin. Alternatively, the tissues
could be synthesizing their own cytokinins and thus have no need for an
exogenous source. Either alternative supports Skoog and Miller's (88)
concept that the balance of horrrones as well as the concentration of
horrrones are critical in determining the direction of rrorphogenesis.
It is interesting that the successful use of cytokinins to produce
whole plantlets has involved the use of B.l\P rather than one of the
naturally occurring cytokinins such as zeatin or 2-isopentenylpurine
(2-iP). B.l\P is relatively cheap ($5.25/g) in comparison with 2-ip ($26.00/g)
or zeatin ($735.00/g), and perhaps this accounts for its popularity.
242

Table 2. Forest tree species in v.nich shoots have been induced t

cuI ture in BI\P.
Angiospenns Gymnospenns

Species Citation Species Citation

Acacia koa 87 eryptaneria japonica 48

-----
Biota orientalis 95 Cgpressus arizonica 95
Eucalyptus ficifolia 7 C. macrocarpa 95
PoEulus alba 30 C. sempervirens 95
P. alba x tremula 30 Picea abies
------ 4, 18, 19
P. anescens 30 -P. glauca 15
P. canescens 17 P. sitchensis 103
P. euroamericana var. Robusta 17 Pinus contorta 103
P. euroamericana var. sat ina 17 P. echinata 14
P. slandulosa 30 P. ell iottii 14
P. nisra var. italica 100 P. J2Cilustris 14
P. nisra var. robusta 18 P. pinaster 34, 35
P. nisra var. tYEica 17 P. ponderosa 14
P. tremula 17, 30, 108 P. radiata 14
P. tremuloides 30, 106, 107, 116 P. rig ida 14
Santalum album 74, 86 P. sabiniana 14
'I'ectona srandis 42 P. s:tlvestris 10, 98
Ulmus campestris 18 P. strobus 14, 68
P. taeda 14, 65
P. vir9:iniana 14
P. wallichiana 59
Pseudotsusa menziesii 25
Thuja occidental is 95
!. Elicata 31
Tsusa heteroph:tlla 26

The reason for the greater effectiveness of BI\P may lie in the
abilities of plant tissues to metabolize the natural hormones more
rapidly than artificial growth regulators. or BI\P could induce
production of natural hormones such as zeatin within the tissues and
 243

thus work through a natural hormone system to induce organogenesis.

Such questions cannot be answered at the present time.

3.4 Other cytokinins

Same authors find zeatin to have either no morphogenetic effect on
cultured tissues or at best find it slightly less effective than SAP
(27, 68, 95, 103). Other authors report strong morphogenetic effects
of zeatin (18, 65, 74, 85, 102). In contrast, Gupta et al. (42) found
that zeatin inhibited induction of shoots on seedling explants of
Tectona grandis. '!hus, the effect of zeatin remains variable even
though it is a natural hormone.
The few cases in which 2-iP has been used in cultures of forest
trees indicated results similar to those found with zeatin. 2-ip has
induced shoots in cultures of Picea abies (3), Pseudotsuga menziesii
(25), Pinus radiata (76), Pinus taeda (65), Populus nigra var. robusta
(18), Pinus contorta (103), and Picea sitchensis (103). Induction of
roots has been reported in cultures of Populus tremuloides (106) and
Pseudotsuga menziesii (28).
2-ip may result in different morphogenetic responses than elicited
by other cytokinins. Winton (106) found that 2-iP induced roots in
callus of Populus tremuloides while SAP induced shoots. 2-ip induced
roots rut zeatin induced shoots in callus of Eucalyptus citriodora (85).
In Ptnus strobus embryos, 2-ip produced only callus rut zeatin or
SAP induced shoots. A related endogenous cytokinin,
6-dimethyladenosine or 2'-isopentenyladenosine (IPA), was found to be
about as effective as 2-ip.
Another synthetic cytokinin, 6-benzyl-9-tetrahydropyrone adenine
(S08339), was found by Rao and Bapat (74) to induce rud formation on
hypocotyl segments of Santalum album. Jacquiot (51) reported that two
relatively weak cytokinins, 6-methylaminopurine and
6-dimethylaminopurine, stimulated growth and induced ruds on callus of
Castanea vesca.
Jacquoit (50) found adenine to be essential for tissue differen-
tiation in a number of hardwoods, rut Risser and White (77) found no
effect on tumors of Picea glauca. '!he presumptive precursor role of
adenine for cytokinins justifies using this compound in aseptic culture
systems.
244

It is clear that a nwnber of cytokinins, roth endogenous and arti-

ficial, can bring about strong morphogenetic effects in cultures of
alnost every forest tree species tested to date. Skoog and Miller's
(88) concept of the cytokinin/auxin balance determining the direction
of morphogenesis has withstood the test of time fairly well and seems
to be valid for a large number of forest tree species.

4. GIBBERELLINS
4.1 Background
Gibberellins are another class of potent growth hormones. They
exert strong control 00 cell elongation and have been associated with
the flowering response. The mechanism of action of gibberellins is
probably 00 cell membranes or at least 00 some cellular component
associated with membranes. Longer-term regulatory effects may occur
through modification of RNA and protein synthesis (102).

4.2. Effects of gibberellins

A few cases have been reported in which gibberellins have enhanced
growth of cultured cells in ooe way or another. Sita (85) found that
gibberellic acid (GA) induced anbryos in callus of Santalum album, rut
Bapat and Rao (6) reported that excised anbryos of that same species
hardly ever developed shoots when treated with G\, although other hor-
mone treatments regularly resulted in plantlets. Strauss and EPP (90)
found that G\ stimulated callus growth of Cupressus funebria. Narasimhan
et al. (72) cbserved an increase in weight of cambial segments fran
Tectona grandis with G\ treatment as well as development of lighter
colored tissue. G\3 stimulated growth of axillary ruds of Eucalyptus
ficifolia (39). Coleman and Thorpe (32) found that G\ inhibited rud
formation in cotyledon explants of Thuja plicata rut later showed that
GA3 and continuous light induced formation of male strobili on callus
(33) G\3 stimulated germination and growth of pollen of Pinus
roxburghii (36). Ie page-Degivery (62) reported that gibberellins
stimulated germination of anbryos of Taxus baccata.
Most authors, OClwever, have reported either negative results or
outright toxicity when gibberellins were used in the aseptic culture of
forest trees. Shibakusa (83) found that Abies sachalinensis shoots did
not respond to GA and also that the native gibberellin-like compounds
 245

in the shoots did rot dlange during imposed donnancy. von Arnold and
Eriksson (3, 4) found ro effect of gibberellins en ruds or embryos of
Picea abies, and Olalupa and D..Irzan (21) found ro stimulation of ruds of
Picea glauca. Similarly, tumors of Picea glauca did rot respond to
GA (77) . Gibberellins had little or ro effect on excised embryos of
Pinus strobus (68) or en cambial segments of Pinus sylvestris (118) or
Pinus monticola (44). Apical neristems of Pinus elliottii did rot
respond to gibberellins (99), ror did ootyledon explants of Pinus
pinaster (34), excised ruds of Pseudotsuga menziesii (1), or callus of
Pinus sylvestris (79).
The failure of aseptic cultures to respond to treatment with gib-
berelin parallels the results of application to intact trees. Except
for inducing flowering in certain species under certain oonditions,
gibberellins elicit little or ro effect when applied to forest trees,
either in vivo or in vitro. FUrthermore, Murashige (70) showed that
gibberellins increased in callus cultures and that they apparently
inhibited norphogenesis (69). We can oonclude that either gibberellins
are rot important in morphogenetic processes or that the endogenous
levels of this hormone are sufficient in most tissues without an
exogenous supply. lance et al. (60) have begun to shed light en this
problem by making careful neasurements of endogenous levels of
gibberellins in culture systems.

5. 0l'HER GRCWI'H-REGULATING SUBSTANCES

A variety of miscellaneous compounds which do rot clearly belong
in any of the categories discussed above have been reported to have
growth-regulating or morphogenetic effects. Al-Talib and Tbrrey (1)
observed stimulation of shoot growth in excised ruds of Pseudotsuga
taxifolia (now ~. menziesii) by tri-iodobenzoic acid (TIBA). Minocha
(68) found that TIBA retarded growth of anbryos excised fran Pinus
strobus seeds rut that it pranoted the fonnation of shoots en the
hypocotyl and en the tips of ootyledons. Similarly, Nanda et al. (71)
showed that TIBA enhanced fonnation of callus and shoot fonnation en
stem cuttings of Populus robusta. It may be that TIBA interferes with
endogenous hormone movement and, as suggested by Minocha (68), there-
fore tends to upset the internal hormone ooncentrations or oon-
centration gradient.
246

Zajaczkowski and Wbdzicki (119) reported evidence for an uniden-

tified compound that stbnulated cambial activity in Pinus sylvestris.
The substance worked synergistically with IAA but was not identified.
Le Page-Degivery (62) found that abscisic acid (ABA) inhibited the
germination of Taxus baccata enbryos, but Il1awan and Malik (36) found
that ABA praroted the germination of tx:>llen tubes of Pinus roxburghii.
Isikawa (48) found a change in pattern of adventitious bud development
in hypocotyl explants fran Cryptaneria japonica \\hen they were treated
with low concentrations of ABA. This stbnulatory effect of ABA is
curious in light of its known close relationship to dormancy (62).
Dhawan and Malik (36) also retx:>rted that ethylene stbnulated
pollen tube growth in Pinus roxburghii. However, ethylene inhibited
axial elongation of embryonic shoots of Abies sachalinensis (83)~ its
reJl'Oval reinstated shoot growth, indicating that ethylene may be par-
ticipating in the regulation of shoot growth in that species. Dhawan
and Malik (36) further reported that adenosine 3: S'-phosphate (cyclic
adenosine nonophosphate or cAMP) stbnulated germination and growth of
pollen of Pinus roxburghii. They concluded that growth regulators
work through cAMP. There is no direct evidence to refute that
viewpoint, but neither is there direct evidence to support it.
Ooumarin enhanced vooting an plumule explants of Pinus sylvestris
(10). The role of coumarin is not known, but it is conceivable that it
interferes with endogenous horJl'Ones or perhaps IAA oxidase and thus
brings about norphogenetic effects (10). It will be interesting to see
if the enhancement of organogenesis by phenolics also applies to other
tree species.

6. CONCLUSIONS
It is clear that growth horJl'Ones playa key role in determining
the course of norphogenesis in higher plants. we have seen in the
above discussion how the various plant growth horJl'Ones produce a wide
variety of effects in aseptic cultures of forest trees. Although
auxins and cytokinins appear to be essential for norphogenesis, we have
seen examples of norphogenesis without the use of exogenous growth
regulators, tx:>ssibly because endogenous levels were sufficient.
Manmade analogs of growth regulators often are nore effective than the
endogenous compounds themselves. The auxin/cytokinin ratio usually
 247

controls the direction of morphogenesis, but not always. TO make sense

of this confusing picture requires that we take into account the
substances endogenous in the systems we wish to manipulate, not just
the chemicals we crld to the rredium. Without such an approach, it is
doubtful that a coherent picture of growth regulator action will ever
emerge.
Several species of forest trees can now be regenerated by aseptic
culture techniques, but the procedures are different for each species.
Techniques effective on one tissue often do not work at all when
applied to other tissues, even from the sarre species. FOr example,
callus derived from cotyledons of Pseudotsuga menziesii can Dorm
shoots, but callus derived from older p:>rtions of the tree and
subjected to those same conditions does not respond (114). Another
cammon observation is that one p:>rtion of a piece of callus may
organize and form shoots but the remainder does not, even though the
entire piece appears to be equally exposed to light and the nutrient
medium. Apparently, endogenous gradients of hormones and perhaps
nutrients can develop in calli to bring about the critical conditions
that induce morphogenesis in one p:>rtion of a callus but not in other
portions. Measurerrents of such gradients have been ~ssible because
appropriate techniques have been unavailable.
Another complication in interpreting the action of hormones is the
interaction between one hormone and another. There is good evidence
that cytokinin biosynthesis may be activated by high levels of auxin
and vice versa (45). Such a response in cultures probably accounts for
the variable hormone requirerrents from one species to the next. TO
complicate the picture even further, different tissues and different
species appear to vary in their ability to produce growth regulators.
Jacquiot (49) found that organogenesis for same species was not depen-
dent upon exogenous growth regulators at all. In view of the large
size of explants he used, there is a strong FOssibility that the endo-
genous regulator levels in those tissues were sufficient to induce
organogenesis. The basic problem which we face, then, is determining
the kind and quantity of growth regulator necessary to supplement the
endogenous ones for each kind of tissue or organ under culture.
The future success of aseptic culture as a rreans of propagating
forest trees depends upon our ability to orchestrate the proper con-
248

ditions, nutrients, concentrations of growth regulators, and tbning of

exposures necessary to achieve the desired response. Without at least
a rudbnentary knowledge of the levels of endogenous growth regulators
in the plant tissues under culture, it will be very difficult to
determine the proper regular supplement without resorting to extensive
empirical trials. Levels of endogenous growth regulators will
undoubtedly change with culture conditions and length of tbne in
culture and may even vary with different explant tissues.
An ~rtant consideration requiring attention is the effect of
nutrients on levels of endogenous growth regulators. Possible
interactions between nutrient and regulator have usually been ignored
when nutrient levels were crljusted. If we are to understand growth
regulator action, b:::Mever, we cannot afford to overlook this
possibility.
Another need is a better understanding of the interaction among
the several endogenous growth regulators and between exogenous and
endogenous ones. The carryover effect of growth regulators, such as
when a tissue or organ is transferred fram a medium of high regulator
concentration to a medium of lower concentration, is not known. This
condition often arises when shoots are induced by exposure to a high
concentration of SAP, then to a much lower concentration to permit
shoot elongation and induction of r<X>ts (28). Not only is the
carryover of SAP in the transferred tissues not known, but the delayed
response of other growth regulators to that high concentration of SAP
has not been evaluated. The effect of 2,4-0 on levels of toth
endogenous auxins and cytokinins could well be a clue as to why 2,4-0
is so effective in eliciting callus production but apparently so
ineffective in inducing organogenesis.
Another possibility is the presence of heretofore unrecognized and
unidentified growth regulators acting in our cultures. There are many
observations indicating the existence of such compounds, but this
possibility should not deter us fram applying what we do know about the
known growth regulators.
The reason why native growth regulators such as IAA and zeatin are
often so ineffective needs to be examined IIOre carefully. Research
along the lines of that reported by Straus and Gerding (91) and Johnson
and Carlson (53) in which IAA activity was related to levels of oxi-
 249

dase reeds to be ~rsued further. '!he role, if any, placed by gib-

berellins in culture systems should rot be ignored, either. Lance et
al. (60) showed that gibberellins occur in tobacco callus cultures, rut
whether they participate in organogenesis has rot been established.
E.W. Weiler (personal oorrmunication) made the intriguing observation
that a sudden rurst of gibberellin production occurred just prior to
the end of the log j:i1ase in suspension cultures of tobacco. Such tran-
sient peaks of hormone activity may account for many of the anomalies
observed in cultures of forest trees. Recent improvements in our
ability to assay small quantities of natural grCMth regulators will
make it possible to determine the levels of these oompounds in tissues
in future experiments. '!he auxin content of sub-gram quantities of
tissue can row be neasured reHably (81, 96 ), and the recessary tech-
niques for neasuring cytokinins in small masses of tissue are being
developed rapidly (104, 117). '!hese technological improvements should
lead to a better understanding of the levels of endogenous grCMth regu-
lators in aseptic cultures in the noreseeable future.
After the historic work of Skoog and Miller (88), plant tissue
cuI ture systems have becx:.me a key tool in research of plant grCMth
regulators. Advances in .o ur understanding of grCMth regulator action
will continue to cx:.me from experiments with aseptic culture because of
the many crlvantages inherent in that technique. Forest trees do rot
appear to differ substantially from herbaceous species in their
responses in tissue culture. With the analytical techniques row
available for regulator assays, our understanding of grCMth regulator
action in forest trees can be improved substantially in the years to
cx:.me. Failure to apply these crlvances in aseptic culture of forest
trees will result in the technique's continuing to be empirical in
nature.
substantial progress has been made in the last ten years toward
producing forest trees by aseptic cultures, and the appropriate use of
grCMth regulators has played a leading role in that success. Future
developrents will depend heavily upon heM rruch we learn about grCMth
regulator action in the years to cx:.me.
250

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Ill. Winton IL (1973) Bibliographic crldendum on tree callus cultures.
Genetics and Physiology Notes No. 18. Appleton, Wis.: Institute
of Paper Olemistry.
112. Winton IL (1974) Second addendum to the bibliography of tree
callus cultures. Genetics and Physiology Notes No. 19. Appleton,
Wis.: Institute of Paper Olemistry.
113. Winton IL (1972) Callus and cell cultures of lbuglas-fir. For
Sci 18:151-154.
114. Winton IL, Verhagen SA. (1977) Shoots from lbuglas-fir cultures.
Can J Bot 55:1246-1250.
115. Withers L (1976) Studies on the growth in culture of plant cells.
J Exp Bot 27:1073-1084.
116. w::>lter KE (1968) lbot and shoot initiation in aspen callus
cultures. Nature 219:509-510.
117. Zaerr JB, Akiyoshi D, MacDonald EMS, ~rris ro (1981) Canbined
high performance liquid chromatography-radiollnmune assay for
cytokinins: A rapid and sensitive substitute for bioassay. plant
Physiol Suppl 67:101.
118. Zajaczkowski S (1973) Auxin stimulation of cambial activity in
Pinus ylvestris. I. '!he differential cambial response.
Physiol Plant 29:281-287.
119. Zajaczkowski S, W::>dzicki ~ (1975) Inhibition and requirement of
natural stimulator for cambial xylem production in isolated stem
segI1l:!nts of Pinus Ylvestris. Physiol Plant 33:71-74.
256

10. NITROGEN METABOLISM AND VEGETATIVE PROPAGATION OF FOREST

TREES
D. J. DURZAN

1. INTRODUCTION
In this chapter, the nitrogen metabolism of cells and
tissues of economically important forest trees is interpre-
ted in the light of our limited knowledge of the specific
genetic gains to be captured by cell and tissue culture
technology.
There are basically two levels to the vegetative
propagation of trees with desired traits. One deals with
organogenesis to regenerate missing parts on cuttings, e.g.,
rooting of needle fascicles or adventitious buds. The other
relies on controlling of the development of cells and
protoplasts for the production of the complete organism
(50). Both have advantages and disadvantages and all
involve nitrogenous compounds.
Emphasis will be placed on the commercially important
coniferous species, Douglas-fir (Pseudotsuga menziesii) and
loblolly pine (Pinus taeda), and on the existing literature
on the suspension cultures of sycamore (Acer
pseudoplatanus). This is only because most information we
have is on these species. The importance of nitrogen
metabolism and the recent advances in molecular biology as
they relate to the propagation of forest trees will be
reviewed. This will be followed by a discussion of nitrogen
metabolism in the physiological and biochemical processes
associated with the control of growth, differentiation and
morphogenesis as seen through nitrogen metabolism.
 257

2. IMPORTANCE OF NITROGEN METABOLISM

In forest soils, nitrogen is the element that often
limits the growth of trees (49, 148). The main form of
rritrogen is reduced, such as ammonium, rather than nitrate,
the typical form in agricultural soils (8, 9). Since the
cost of applying fertilizers is becoming increasingly
prohibitive, trees that are more efficient in utilizing
nitrogen or that are nitrogen fixers should be selected and
cloned (16, 17).
Nitrogen contributes to the building blocks of cells:
to proteins, nucleic acids, and enzymatic co factors that
catalyze the conversion of substrate to product. Many of
the natural and synthetic growth regulators supplied to
tissue culture media contain nitrogen or in one way or
another affect the events in nitrogen metabolism (e.g., (3-
indole acetic acid, IAA; N6 -benzylaminopurine, BAP).

2.1. Range of naturally occurring nitrogenous compounds

in forest trees. Historically, glutamate dehydrogenase
found in cell membranes has been regarded as the enzyme
accounting for nitrogen assimilation. Now this port of
entry is thought also to be mediated by the GOGAT enzyme
(Fig. 1). In both systems, glutamate is an early product of
nitrogen assimilation. Alternative reactions accounting for
the entry of nitrogen may yet be found (59).
Once nitrogen enters glutamic acid and glutamine, all
the other 20 or so amino acids found in protein can be
formed. This is accomplished by nitrogen transfer: the
transamination of the glutamate with the appropriate carbon
acceptor (Fig. 2). The elaboration of nitrogenous compounds
is not restricted to the protein amino acids and involves
the wide range of free amino acids encountered in plants
(59). Furthermore, once amino acids are bound in protein,
at least 140 different post-translational modifications can
add to structural and functional diversity (160, 172).
 258
LIGHT

Hl
CHLOROPHYLL

"'T' . . . .---- ferredoxin-

_---_-~r~~Oin 2
thioredoXin .....
reductase _ ~
~ 10
2

enzyme control
.... " H~
.......... " ATP ADP
ferredoxin .;
_~-:-_".~I N02-----~---- -..,.-l""'--'-_ glutamine
nitrate nitrate
reductase reductase

CYTOSOL

ferredoxin
glutamate synthase

a-ketoglutarate

CHLOROPLAST transaminase

"- a-keto acids amino acids

~
PROTEIN

KEY

I Primary N acceptor I
Product of reaction

FIGURE 1. Primary port of entry for nitrate nitrogen into

organic combination in green leaves in the light. Nitrate
is reduced to ammonia (NH 3 ) which enters the reactions
mediated by enzymes of the glutamine synthetase-glutamate
synthase system. This produces the first formed organic
nitrogenous product viz. glutamine (glutamine synthetase).
The acceptor for the reduced nitrogen is L-glutamic acid.
The glutamine then reacts with o(-ketoglutarate to produce
 259

two equivalents of glutamic acid (glutamine [amide] ) :

2-oxoglutarate amino transferase [oxidoreductase NADP]. One
of the glutamate molecules continues to incorporate reduced
ni trogen. The other contributes through transamination to
the formation of amino acids commonly found in protein.
Other ports of entry may exist in plants, especially in other
organs and in vitro. Under high levels of reduced nitrogen,
glutamate dehydrogenase is c onsidered to be the enzyme at the
primary port of entry for most plant tissues. Glutamic acid
then becomes the first organic nitrogenous compound formed
(59) .

Free and protein amino acids may be categorized into

families depending on the first amino acid in a biosynthetic
sequence (Fig. 2). The aromatic family (phenylalanine,
tyrosine and tryptophan) contributes to the formation of
auxin (indoleacetic acid) and its conjugates. Members of
the aspartate family produce ethylene, probably via l-amino-
cyclopropane carboxylic acid. Mevalonate, produced from
carbon frameworks lacking nitrogen, leads to the formation
of gibberellins (GA) and abscisic acid. In some germinating
seeds, GA are believed to regulate protein turnover (162).
The remaining growth regulators, especially the cytoki-
nins, seem to arise from the turnover of nucleic acids,
although evidence is increasing for their de novo formation.
The nucleic acids, being composed of purine and pyrimidine
nucleotides, derive their nitrogen from amino acids (59) or
from purine or pyrimidine supplements to culture media. The
nucleic acids are normally produced de novo from the simpler
building blocks, such as from the amino acids, or from the
salvage of nucleotides, purines, and pyrimidines generated
by metabolic turnover.
The wear and tear of cellular activities is repaired b y
enzymes through sustained turnover of macromolecules (59).
Turnover contributes to the compartmentalized pools of free
nitrogenous compounds in cells. Protein turnover contributes
the amides, o{-amino acids and their secondary products such
as the amines and unusual free amino acids. The nucleic
acids contribute the purine and pyrimidine nucleotides,
260

TRYPTOPHAN AUXIN
TYROSINE
PHENYLALANINE
HISTIDINE
C02---91~Y1--1I.-ta--------P_h_ot_oS~y_n~--_is--------------~F-~~ ~
'glutamine
t
glyo.ylate
TTP
pentose phosphata path

6
~ erythrose-4-phosphat8

"'-A--U---N-IN--'E
> ~:~~7~E
glYCOlYSiS!

GLYCINE ~S~~~uhv~:O.y- ~ /\, N

~~~~~~NE ~Pyruvata
TTP
~

<
NAD+ CO 2
CoA

CN- I NA~~+ I
Acetyl CoA IC 2 fragment)

J
ASPARAGINE
>
A~!~~TATE \ ___N_A-'--~-'--H_+H--;-+~o.alo.cetata IC4 ) IC 6)
Citrata\
KREBS CYCLE

HOMOSERINE*
I -J NAD+
t C, t '-----.
=,~~~',~i ."",0",,, -\ "_m ..~:*:

Fumarata a-Ketoglutarate IC S) fNl

Reduced flavoprotein..
.
~ Succinata ~- A ~ ~
' - CO 2 N CARBAMYL
flavoproteIn /C4) PHOSPHATE

SuCCinic-~-,?
N
..mialdehYddl GLUTA~111
GLUTAMINE I
~-AMINOBUTYRATE ARGININE
CO2 PROLINE

FIGURE 2. The formation of 20 protein amino acids and

n-aminobutyric acid by nitrogen-transfer reactions and
carbon derived from sucrose in the medium, photosynthesis
and respiration: families of amino acids. Nitrogen in
glutamic acid or other amino acids may be transferred to
carbon acceptors to produce a wider range of families of
free amino acids. Transfer points for nitrogen are designa-
ted by arrows containing the symbols Nand CN- (cyanide) .
The first amino acid synthesized in the family is printed in
large type (e.g., ASPARTATE). Secondary amino acids,
derived from the first one synthesized, are shown within
each box. Sucrose and thiamine (TTP) , added to the culture
medium, are believed to influence the rates of biosynthesis
of nitrogenous and carbon compounds at the points illustra-
ted. Photosynthesis, sucrose in the medium, and the Krebs
cycle generate energy as ATP (adenosine triphosphate) to
 261

drive the reactions as substrate are oxidized (2). In each

family, ATP is needed in different amounts to synthesize the
amino acids.

A
II I---
-tm>KtN~"':=~N--:- -- -- -- -- -- -- - "\
i~ threshold of integrated correlations

I
I
1 '
NUCLEOTIDES
~s
~
ENZYME FORMING SYSTEM .C ~i:

I
=t I
INDUCTION--+...!,..+---- REPRESSION

E N Z Y M E " DEGRADATION
(PRIMARY AMINO
~ACT'VATOON~ t--'NH'."'ON "'0'1

_4i)IaH,"BS,"m ../ 7lW0i8'AC'OSI II

I f I

MEVALONATE / AROMATIC, GLUTAMATE, ASPARTATE, ALANINE, GLYCINE ,

OA
I
A$$CISICACID

1- -d~I!li~.~~.c pt_ -1;.[llllt-- - - _./
FIGURE 3. The relationship of enzyme-forming systems
originating from information in DNA to nitrogen assimila-
tion, to the conversion of substrate to amino acid products,
and to the production of certain plant growth regulators.

ureides, urea, and the ~ -amino acids to the pool of ni tro-

genous compounds.
These substrates and products may control the enzyme-
forming system through inducing or repressing enzyme synthe-
sis and by activating or inhibiting enzymatic activity. The
cyclic nucleotides are believed to control the phosphoryla-
tion of key proteins during cellular development. Evidence
is rapidly accumulating that shows that many enzyme systems
are modified by macroelements such as calcium and potassium
or by special proteins such as calmodulin and ATPases (91).
The intermediary metabolism of nitrogenous compounds
and the turnover of macromolecules through controlled
enzymatic activity leads to the production of a wide array
of chemical homologues, analogues, cyclic substitution
products (59), and endogenous plant growth regulators (Fig.
3) . These, in turn, progressively determine and control
growth and development through physiological thresholds
262

based on an integration of correlations in the explant or

propagule as a whole (133). These biological and chemical
thresholds are required to launch development. Other
thresholds may be satisfied by temperature or light of
specific wavelengths (154).
In jack pine cells and callus, the composition of the
free amino acid pool changes sequentially during overall
growth (52, 53, 54, 55). Nitrogen for this metabolic
sequence is derived from the medium and in cells by a drop
in protein level.
Ni trogenous compounds are
commonly identified by the
structure of the nitrogenous moiety (59). For example,
conifers are sometimes rich in monosubstituted guanidines,
which are derived metabolically from the parent amino acid,
arginine (54). In studies of nitrogen metabolism of the
whole tree, the ureides, amides, arginine, and monosubstitu-
ted guanidines have usually received the most attention.
These nitrogen-rich compounds serve as storage and translo-
ca ted forms of nitrogen and even may inhibit growth and
development.
Synthetic growth regulators containing nitrogen, such
as benzylaminopurine and zeatin, are used to control growth
and morphogenesis in vitro. These substances may form
conjugates with amino acids (Table 1). Conjugates remove
physiologically active compounds and may be very effetive in
controlling plant development, especially if the active form
is released later (78, 100).
In view of the wide diversity of nitrogenous compounds,
the determination of developmental events in cells of woody
species should relate eventually to the polymorphism and the
vast number of posttranslational modifications of proteins
for the control of enzyme activity and to the processing of
nucleic acids during replication, gene expression,
transcription, and cell transformation. However, because of
the rich tannin and phenol content of tree tissues, the
study of macromolecules was set back initially because of
binding artifacts with proteins. The situation was improved
 263

Table 1. Exalrples of amino acid conjugates of natural and synthetic plant gra.>t:h regulators.

Narre and Structure CXcu.rrence, Recognition, etc. References

N- (inoole-3-acetyl) aspartic PD:::>ts of "alaska" peas 1955 WA Andrae and

acid II E Good, Plant
Physio1. 30:380.

Seeds of several plants 1960 H D von Klilrrbt,

Natw:wiss, 47:398.

6-L-lysine conjugate in Pseudrnonas 1968 0 Hutzinger and

T Kosuge, Biochem.
7:601.

Aspartate, glutamate glycine, alanine 1977 COS Feung et

and valine conjugates in Crown-gall a1., Plant PhysioL
callus of Parthenocissus tricuspidata 58:666.

a -Naphthalene aoetic acid .6 -D-Glucose and aspartyl conjugates in 1979 J Riov et a!.,
conjugates Pinus sp. Physio!. Plant 46:
o

O)'CH2-CII-R
133.

5 rretabolic products deter4ed in jack D J Durzan and R A

pine cell suspension fed C-Naphthalene canpbell (unpub-
acetic acid lished)
R-conjugated noiety

N- (2, 4-Dichlorophenoxyacetyl)- Soya bean plants 1971 D I Chkanikov

L-aspartic ~cid et a1., Fiziol. Rast.
19:436.
Conversion to N-(2-nethyl-4-chlorcphen- 1970 D J Collins and
oxyacetic acetyl) -L-asparate J K Gaunt, Biochem.
J. 118:54.

L-<;lutarnic acid conjugate 1971 C S Feung et a1.,

J. ~r. Food Chan.19:
475.

2,4,5-Trichlorophenoxyacetic Soya bean callus 1978 M Arjamand et

acid derivatives al., J. ~r. Fooi:f"
cnem. 26:1125.

Phaseolus vulgaris shoots 1979 D S I.etham et

~., Planta 146:71.

Conjugates in cells and callus in true 1980 R Feirer, D J

ent>ryos of Douglas-fir Durzan (unpublished).

when phenolic compounds could be removed during the isolation

of enzymatically active proteins. Further improvement was
obtained with the control over nucleases and proteases that
degrade chromatin.
The nitrogen content of cells serves as a baseline for
judging metabolic and developmental events. Since several of
the macroelements and trace elements in nutrient formula-
264

tions become part of metalloproteins, changes in macroelement

composition of cells are often best described on a protein
basis. Metalloproteins undoubtedly affect the growth and
metabolism of cells and tissues (82).
2.2. Gene expression and mapping. The mode of gene
expression remains to be investigated (14, 50, 67, 130,
138). Coniferous species have more DNA than other plant or
animal species (117). During the replication of DNA, the
nitrogen in bases composing DNA is derived from the amino
acids and passed to daughter cells.
Fragmentary studies have indicated that the process of
gene expression involves transcription of DNA and transla-
tion of the information in DNA into specific proteins. This
process appears to be the same for most higher organisms.
During organogenesis, levels of Feulgen-positive material
(DNA) in chromosomes seems to be amplified (112). Cellular
differentiation (21), vegetative reproduction, and apomixis
(76) are correlated more with chromosomal changes and ploidy
levels, but in different ways (Figure 4). This process is
not clearly understood.
For coniferous trees all known components of chromatin
have been isolated and examined (113 to 116). Chromatin
includes DNA, histones, and the nonhistone chromosomal
proteins. The replication of DNA in forest trees seems to
follow models proposed by Lark and Cress (96): the DNA is
replicated in short units called Okazaki fragments (108).
The control and processing of gene activities will
eventually have to relate to the elimination, amplification,
rearrangement (directed or nondirected) and modification of
genes in the nucleus and in organelles of the cytoplasm. We
will have to understand how gene traits are controlled,
processed, scheduled, and transmitted during the life cycle
and how stable these traits will remain if introduced in
vitro.
The availability of a gene map (and an understanding of
how nitrogen contributes to growth and development) should
facilitate research work considerably. It is important to
 265

RIBONUCLEOTIDE
REDUCTASE
(THIOREDOXINI

CHROMONEMA R"'E.'"'LlCA=T'":':ON'1
f':1 -1 % HETERO-

ii
NUCLEAR

!e
RNA
CHROMATID ORGANIZATION
(mRNA
PRECUAmRt

1i1l
!~

CENTROMERE DtVISION
CHROMOSOME I REPLICATION I
/
CYTOKINESIS
.mM
POLAR MIGRATION OF DAUGHTER CHROMOSOMES
CEllI REPLICATION I
NUCLEAR I REPLICATION I
A B
FIGURE 4. Nitrogen metabolism and the mitotic cycle in
higher plants (A) relate the behavior of DNA in chromatin to
the replication of cells. Daughter cells contribute to the
life cycle of the tree and to apomictic or sexual (meiosis)
reproduction. The stages of the cell cycle are Gl, S (DNA
synthesis), G2, and mitosis. B depicts a single strand of
chromatin and shows how and where messenger RNA is believed
to be formed. The integrity of the strand of chromatin is
shown as a function of increasing ionic strength. C empha-
sizes the flow of nitrogenous compounds via ribonucleotide
precursors into deoxyribonucleotides for the synthesis of DNA
and into ribonucleotides for the synthesis for all types of
RNA. However, in cytokinin-directed organogenesis, DNA in
cytochemical studies seems to have several fates. DNA levels
can be reduced, and sections can be eliminated, amplified,
rearranged, and modified. Changes in DNA levels during the
organogenesis of Pinus coulteri have been described by Patel
and Berlyn (112).-----

know whether ideal traits are representative of the linkage

groups of the genome, and it is also useful to have markers
on the chromosomes so that genes or specific characters and
their metabolic products can be mapped. If economically
important traits are inherited in blocks, gene markers
linked to them would be of great value for selection pur-
poses. Here the diversity or polymorphism in chromosomal
proteins and nucleic acid transcripts of DNA will become a
significant aspect of the progressive genetic determination
266

of the physiological state of the cell and the control of

morphogenesis.
Gymnosperm seeds are especially suitable sources of
tissues for propagation and for electrophoretic analysis of
gene populations. Without breeding experiments, we can
compare the nucleic acid and protein patterns in cells of
the maternal haploid with the diploid tissues of the prog-
eny.
Electrophoretic analysis of nucleic acid and protein
variation (45) is an efficient method of determining genetic
variation at a large number of gene loci (e.g., 7, 126,
170) . For example, using polyacrylamide gel electrophor-
esis, Bartels (7) demonstrated the genetic control of
mUltiple esterases in needles and the female gametophytes of
Picea abies. Isoenzymes, however, reflect transcriptional
and translational events after they have occurred.
For developmental problems in vitro, the polymorphism
of macromolecules and the unscheduled synthesis of DNA will
have to be considered in more detail, especially in relation
to the action of auxins and cytokinins (118). In haploid
and diploid conifer tissues, Pi tel and Durzan (115, 116)
point to the nonhistone chromosomal proteins (NHCP) and the
polymorphism of chromatin as important elements contro.lling
gene expression.
An intuitively appealing approach to recognizing new
developmental patterns of cellular behavior in vitro in-
volves DNA or RNA template matching. However, selecting a
basic representative template for the different classes of
patterned cell development may be difficult.
2.3. Metabolic changes in organized and unorganized
systems. The physiological state of explants is a key
factor affecting nitrogen metabolism during in vitro propa-
gation (51).
The state may be described at five levels:
1) A description of the genotype and phenotype as a
function of the explant source. This provides a
 267

perspective for understanding the specificity of

changes in nitrogen metabolism.
2) Proof of the heritable nature and mode of trans-
mission of traits under consideration. For
example, it is important to understand if genes
are eliminated, amplified, rearranged, or modified
during development.
3) Identification and quantification of any special-
ized metabolism including abnormal metabolites,
critical levels of primary and secondary metabo-
lites (indoles, methylated purines, amino acids),
and the sequential appearance of storage substan-
ces during morphogenesis.
4) Demonstration of a defective or altered enzyme or
structural protein creating problems or a unique
set of activities accounting for new traits.
5) Identification of the mutational or genetic event
that alters the protein (s) and the reasons the
event is not corrected. This includes the need to
recognize epigenetic factors (18).
In the cell, changes in nitrogen metabolism may be
easier to detect in vitro than in vivo. Cell suspension
cultures offer the following advantages:
1) Only a few cells are needed as starting material
to establish the culture.
2) The growth of cells in liquid medium can be better
attuned and scaled-up under defined nutrient
condi tions by synthetic and natural plant growth
regulators.
3) A uniform population of cells or tissues can be
produced, and in some cases their growth and
development can be synchronized by environmental,
physical and chemical factors.
4) The nitrogen metabolism of cells in vivo and in
vitro can be compared with similar events in the
donor tree.
268

5) Model cellular systems (e.g., carrot and sycamore)

are available for comparison with cells in suspen-
sion culture (161). For example, in wild carrot
embryogenesis, nitrogenous compounds (arginine,
polyamines, and conjugates of growth regulators)
(100) are believed to control development.
6) The space to grow cells and develop plants is
usually less and better controlled than most other
culture systems.
7) If biochemical markers for elite traits are known,
large populations of cells can be screened for
these traits. Individuals can be selected direct-
ly or indirectly, and if needed, preserved
cryogenically.
8) The fate of isotopic tracers can be evaluated in
homogeneous cell populations free from contamina-
ting organisms, complex structures, and correla-
tions and under a wide range of environmental and
nutritional conditions.
9) Cells in suspension cultures may experience
partial or total rejuvenation. The rejuvenation
is due largely to the removal of constraints
imposed by neighboring cells, meristems and
metabolic products commonly found in the translo-
cation stream. With mature tissues rejuvenation
remains a serious problem.
10) In suspension cultures, the metabolism of cells
and organized cellular masses can be modified by
controlling of substrate and product levels. In
some cases metabolites may be produced commer-
cially (137).
11) Protoplasts can be prepared from cell suspension
for the application of genetic engineering tech-
nologies. Fused protoplasts containing multiple
nuclei of the same or different genetic source
(haploid or diploid) can be constructed and
screened by size of the fusion product. Since the
 269

zygote passes through a multinuclear stage,

comparisons with protoplasts with multiple nuclei
regarding the effect of such genetic loads on the
developmental fate of cells may be sought.
12) With gymnosperms, mutations can be introduced in
haploid cells. Mutations are not masked and are
expressed directly. For example, Tulecke (156)
has found haploid cells that require arginine.
Homozygous diploids and hypohaploids could be
induced from haploid cells (13, 111, 157).
2.4. Nitrogen and nutrition. Supplements to the nutri-
ent medium that foster better nitrogen nutrition require a
careful choice of carbon source (cf. Fig. 2) . The most
suitable carbon source allowing cells to produce the appro-
priate carbon acceptors for amino acid production is su-
crose.
Compounds more closely related to the actual point of
entry of nitrogen, such as the~-keto acids, are unstable in
aqueous solutions, toxic to cells, and expensive. In many
cases they are unsuitable as sole carbon sources in nutrient
media.
Ammonium salts, nitrate, urea, the amides, ureides and
some amino acids have been found to be good nitrogen sour-
ces. Growing cells absorb ammonium over and above their
ability to form protein (38). Should the GOGAT system (Fig.
1) operate, ammonium assimilation will consume ATP. By
contrast, organic forms of nitrogen in the medium, if
assimilated, may spare some of the energy (ATP) normally
used for the entry of nitrogen into organic combinations
(2)
Conifers are sensitive to ammonium, so care must be
taken to ensure against toxicity. For this reason, nutrient
formulations should contain low levels of urea (e.g., 47,
48, 124 and Durzan, unpublished). Amides and ureides are
tolerated at higher levels than ammonium.
One tendency has been to load media with products of
protein and nucleic acid breakdown, such as casein hydro-
270

lysate, amides, purines (adenine sulfate), pyrimidines,

ureides, and r-amina acids. These campaunds are faund in
the nutrients surraunding the natural embrya as it cansumes
the female gametaphyte during grawth. A carnman error is ta
supply N-rich campaunds in taa high an initial
cancentratian. An autatraphic habit can sametimes be
encauraged by a shift in the farm .of nitragen (e.g., fram
urea .or NH4 + ta NO;), by high light intensity and by the
remaval .of vitamins .or grawth regulatars (57).
Anather appraach ta determining nutri tianal needs .of
cells is ta recanstitute the knawn nutrients .of the develap-
ing seed (65). Here the nitragen is simple and reduced
+
(e.g., NH4 .or urea). Catians .occur as simple .organic salts
and rarely as nitrates In recanstituted nutrient media,
.organic salts are .often beneficial (e. g., K, Mg and Fe
glyceraphasphate) (DJ Durzan unpublished) . Cells .of
Dauglas-fir in suspensian are quite talerant .of recanstitu-
ted media with law levels .of inarganic salts, especially law
calcium: this seems ta be true far same but nat all
species. Far example, white spruce prefers high levels .of
calcium, particularly calcium nitrate (60). The recently
discavered calcium-cantaining pratein calmadulin, naw faund
in same plants (91), shauld be saught in waady species.
In cell suspensians .of canifers, arginine has always
played a praminent rale in ni tragen metabalism; Table 2
illustrates the influence .of arginine and its interactian
with grawth regulatars.
Grawth regulatars in the medium pramated the grawth .of
cell suspensians at the expense .of amina acids in cells.
The remaval .of supplemental arginine in the presence .of
auxin and cytakinin impraved the yield .of cells and resulted
in a small paal .of saluble N campaunds ~males/g fresh wt).
The additian .of an arginine supplement inhibited the pralif-
eratian .of cells; the inhibitian was .overcame by the addi-
tian .of synthetic grawth regulatars. This invalved a
reductian .of amides (glutamine and asparagine). With the
remaval .of arginine, the mast respansive amina acids in the
 271

presence of growth regulators were -aminobutyric acid,

which accumulated to 17.5;umoles, and serine, which declined
to 4.5 }-lffioles. Studies with arginine inevitably lead to the
recovery of amines (e.g., ornithine, agmatine related to the
glutamate family of amino acids) (Fig. 2, Durzan and Lop-
ushanski unpublished).

Table 2. Free amino acid composition of Douglas-fir cells

grown in suspension and in darkness for 30 days. The medium
(1/4-strength Murashige-Skoog 104) was supplemented with
arginine (400 mg/liter) and growth regulators (2.5 mg/liter
NAA, and 0.1 mg/liter BAP) as indicated (+). Data represent
the amino acid composition on a mole % basis (unpublished,
preliminary data courtesy Institute Paper Chemistry).

- Arginine + Arginine

- Growth + Growth - Growth + Growth

Amino Acid Regulators Regulators Regulators Regulators

asparagine 28.3 26.4 27.8 3.5

aspartic acid .7 2.2 1.5 1.2
threonine .2 .8 .1 1.1
lysine .1 .3 t .5

glutamine 38.7 11. 7 56.1 12.6

glutamic acid 4.5 16.1 3.3 11. 4
proline 6.8 1.2 4.5 1.2
(-aminobutyrate 1.3 17.5 .5 17.2

alanine .8 4.6 .4 13.3

leucine .2 .4 .1 .8
isoleucine .3 .4 .1 1.3
valine .8 .3 .4 1.4

serine 13.2 4.5 1.1 4.7

glycine .6 .3 .2 .2
methionine 0 0 0 0
tyrosine .1 .1 0 .2
phenylalanine .5 .2 .2 .4
histidine .5 .1 .1 .2
tryptophan .5 .1 0 .2
cysteine 0 0 0 0
arginine .2 4.2 4.5 21.6
total pmoles/
g fresh wt 9.2 8.3 64.3 12.3

cell yield/flask
(grams by wt) 0.29 9.11 0.18 7.13
272

Concerning the synthetic amine EDTA, Singh and Krikorian

(131, 132) have drawn attention to errors associated with
the chelation of iron in the formulation of nutrient media.
The excess EDTA is thought to bind other trace elements,
such as zinc and copper. However, the physiological effect
of this amine at cellular binding sites such as membranes
and chromatin cannot be ruled out and requires further
investigation.
2.5. Aspects of intermediary nitrogen metabolism. This
section deals briefly with the fates of reduced forms of
nitrogen, particularly ammonium salts, urea and purines,
because these substances are common supplements to culture
media. In model systems they are required for somatic
embryogenesis (166). In cells, endogenous urea can origin-
ate from the storage compound arginine or from the degrada-
tion of purine supplements. Since plants tend to conserve
their nitrogen, the nitrogen of urea, which rarely accumu-
lates, re-enters metabolism largely as glutamine, aspara-
gine, and carbamyl phosphate. The latter involves the
formation of carbamates and nitrogen transfer usually from
glutamine (59).
The simpler forms of organic nitrogen, such as urea,
and the key metabolic intermediate, carbamyl phosphate, have
been studied using carbon-l4. With white spruce callus,
derived from hypocotyls, the fate of 14C-urea is shown in
Fig. 5. Conspicuous products with carbon derived from urea
are alanine, serine and glycine consistent with pathways
shown in Fig. 2. The metabolites originate through the
action of urease to release 14 co2 , which is then refixed by
photosynthesis. Another noteworthy product is carbamyl
aspartate, which is a precursor for pyrimidines. Products
of urea metabolism are widely dispersed through intermediary
metabolism and are recovered from proteins and nucleic acids
(47,48,59).
The metabolism of 14c-carbamyl phosphate is complicated
by cyanate metabolism because of the instability of the
former in aqueous solutions. Nevertheless, with proper
 273

3S

.....
CD

..
~
CD

.
U

..
"0
c
:J
,g
2

31
phenol

FIGURE 5. Autoradiograph of a paper ch1:.~matogram revealing

the radioactive metabolic products of C-urea applied to
the top of a white spruce callus derived from hypocotyls and
grown on the medium. of Durzan et al. (60). Each gram of
callus in the fourth subculturewas-extracted for metab~!ic
intermediates after 30 minutes of exposure to 10 !-'Ci C-
urea of high specific activity. Tentative identifications
are based on color reactions and cochromatography in two
systems. Other identifications are supported by similar
criteria on an automated amino acid analyzer (physiological
fluids, Beckman 120C). Key to the identity of radioactive
spots in Figures 5 and 6: 2 asparate, 3 glutamate, 4
serine, 5 glycine, 6 asparagine, 7 threonine, 8 alanine, 9
glutamine, 10 carbamylaspartate, 13 arginine, 23 Y-amino-
butyrate, 112 ethanolamine (tentative), 118 citrulline, 119
argininosuccinate (tentative), 142-143 products related to
biotin. All others are unidentified.

controls, the immediate products common to urea metabolism

were carbamyl aspartate, glutamine, and ethanolamine (Fig.
6) . Carbamyl phosphate contributed to the formation of
citrulline, argininosuccinate and traces of arginine within
the short exposure time. Results with callus on agar plates
are consistent with metabolic pathways involving the urea
cycle in seeds and trees (59, 106).
274

142

143

urea ...
...
Q)

III
~

23 112 ...<i
Q)
8 10
(J
III
3
0
c

118
4 ...::s
III

9 119 .Q
13

phenol , e.J
FIGURE 6. _Autoradiograph of a paper I~romatogram revealing
the radioactive metabolic products of C-carbamyl phosphate
applied to the top of a white spruce callus as in Fig. 5
(Durzan and Lopushanski, unpublished). The l~allus was
extracted after 0.5 h of exposure of 10 ~Ci/g C-carbamyl
phosphate. Carbamyl phosphate is the large chromatographic
spot at the extreme right and moving in the direction of
acid butanol. Other unidentified products are associated
with this spot. Key to the identity of radioactive spots is
the same as in Fig . 5.
When cytokinins or purine derivatives are added to
medium, the pattern of N metabolism is usually significantly
altered . Purines have several fates, including degradation
to urea and salvage of the products for nucleotide synthe-
sis. Purines also have physiological effects that may
influence morphogenesis (14,59).
As for auxins, Riov et al. (122) found that incubation
of tissues of Pinus ~. with NAA_1_ 14 C resulted in the
formation of l-Q-naphthylacetyl-~-D-glucose and ~-naphthyl
acetyl aspartate . Unpublished work at The Institute of
Paper Chemistry indicates that similar conjugates of N6 _
benzylaminopurine are formed in cell suspensions and in
cultured excised embryos of Douglas-fir.
 275

3. NITROGEN METABOLISM IN GROWTH AND DEVELOPMENT

In simplest molecular terms, cells, callus or tissues
grow if DNA molecules replicate and repair themselves and if
the rates of RNA and protein synthesis are greater than the
corresponding breakdown rates (59, 121). During growth,
nitrogen assimilation contributes to the net increase of
macromolecules and cellular components. This flow of
nitrogen is important for increases in cell size and numbers
of cells. The initial allocation of nitrogen to specific
compounds, especially nucleic acids, new proteins and
storage products is performed by organized enzymatic net-
works and by different cells in multicellular systems (Fig.
3)
Morphogenesis occurs when information in DNA is sched-
uled for the formation of new subcellular systems, cells,
organs and individuals (59, 67, 138).
Every culture system has a number of general proper-
ties, such as maturity, nutrient inputs, responses to growth
regulators, and developmental potentials. Any combination
of factors is often referred to as the "physiological state"
of the system. The tissue culturist faces a choice of which
tissue is in the best physiological state for propagation
and what factors best contribute to normal completion of the
life cycle (51, 125). To help provide the background,
Appendix I presents an annotated and chronological summary
of selected references to studies of nitrogen metabolism
performed with cells, tissues or excised embryos in vitro of
gymnosperm and angiosperm tree species. Most reports,
however, deal with the role of nitrogenous compounds of low
molecular weight rather than the control of basic develop-
mental events.
3.1. Precultural factors. For woody species with their
perennial habit and intermittent growth, considerations that
are always important are the heredity, physiological state,
polarity of auxin flow, and composition of the tissues at
the time of selection (34, 51, 73, 111, 112, 171). When
tissues are placed in vitro, the hope is that the physiolog-
276

ical state at the time of excision does not hinder subse-

quent attempts to control development .
One example of the wide variation in composition of
explants is the seasonal and diurnal changes in free amino
acids of spruce buds. When resting buds rich in free
proline are excised and placed in vitro to promote shoot
elongation, the emerging shoots recapitulate the typical
sequential changes in amino acid composition found in buds
under field conditions (28, 29). Moreover, when excised
tissues form new buds under the influence of synthetic
growth regulators, nitrogenous compounds, such as arginine
and the monosubstituted guanidines, accumulate and charac-
terize each stage of development.
The tree may be pretreated with growth regulators
before explants are selected. In these situations,
nitrogenous storage compounds are converted to amides:
blocks in metabolism inhibiting morphogenesis are removed,
and a more responsive explant is produced.
With trees of proven value, the simplest molecular
approach is to search for the least mature tissues rich in
DNA, RNA, indoles, arnides, and amines found at meristems
(buds) or to use juvenile shoots formed at the bole of trees
(e.g., Sequoia, 3). Other sources of nitrogen-rich cells
are the tips of needles, the cambium, and tissues from
developing seeds. A stock of juvenile or reinvigorated
older shoots can be maintained if the species is responsive
to the practice of hedging and to the addition of nitrogen-
ous fertilizers. Hedges are grown for the production of
numerous young shoots for cuttings that are then rooted.
Twigs or seedlings may be grown on agar and pretreated
before explants are taken. The pretreatment, be it chemical
or biological (e.g., grafting onto juvenile rootstock),
"conditions" or invigorates the tissues for the next step of
the in vitro process . The initial steps should permit
screening of tissues against contamination and pathogens,
such as virus, mycoplasma, and bacteria.
 277

Through nitrogen metabolism, explants may be condi-

tioned by growth under different environmental (e.g., long
days), nutritional (enriched media and CO 2 ) and growth
regulator regimes (auxins, cytokinins, gibberellins, etc.)
a s we 11 as by factor s that remove growth inhibitory com-
pounds, such as phenolics, abscisic acid, guanidines and
their derivatives. Removal is facilitated by adding to the
medium of peroxide, mercury, charcoal, and polyvinylpyrroli-
done. In some instances, phenylamines, as contaminants or
reaction products in charcoal, stimulate morphogenesis (26).
Aldehydes and ammonia are reaction products of amino acids
with hydrogen peroxide (173).
Once the explant is aseptic and the past development of
the tissue is understood in terms of cytogenetics and auxin
distribution, metabolism, and polar transport, the induction
of adventitious buds or callus formation is performed,
usually on agar. The suitability of a propagation system
depends on the ease of establishment of cell suspension
cuI tures and on organ or embryo regeneration. The most
advanced method currently is plant regeneration through
organogenesis directly on the explant and without the
intermediary formation of callus (David, this volume).
3.2. Callus formation. Some scientists believe that
genetic instabilities arise during callus formation (81,
Ill, 112). This instability may be minimized by exposing
cells to growth regulators at low levels 2 ppm) or at high
levels for short periods for callus initiation. If genetic
instability is related to the loss of biochemical informa-
tion in macromolecules, then control over the activity of
enzymes such as proteases, nucleases, repair enzymes and
nonhistone chromosomal proteins should be sought. Indeed,
with some somatic cell lines and especially with cell
suspensions of Douglas-fir from 50-year-old trees, results
in early stages of morphogenesis have been encouraging after
a protease inhibitor has been added to the nutrient medium
(Hwo and Durzan, unpublished).
278

Reduced and oxidized forms of nitrogen seem sui table

for callus production with NH 4 N0 3 being one of the more
commonly used forms. Ca(N0 3 )2 provides additional benefits
in white spruce but not in Douglas-fir (60). The key to
controlling production of cells in callus is to provide
continually a balanced nutrition under the influence of
auxins such as c(-naphthalene acetic acid or ;3-naphthoxy-
acetic acid. The rapid proliferation of cells disrupts the
momentum of past patterns of growth and leads to cellular
enlargement, which must be limited. Too much enlargement
reduces the totipotency of cells and often indicates genetic
instabilities.
Other aspects require special attention at the callus
stage: 1) Low levels of chemical contamination, e.g.,
biuret in urea, pyroglutamate in glutamine, and D-amino
acids in L-amino acid preparations need to be controlled.
2) Excessively high levels of nutrients may lead to the loss
of totipotency with the appearance of unusual pathways of
nitrogen metabolism. 3) Long-term and high-level growth-
regulator requirements should be evaluated in case of
unexpected habituation of callus to these substances. 4)
Somatic mutations, such as loss of chlorophyll or reversion
to unusual growth patterns, should be closely monitored. 5)
Latent microbial contamination, especially masked by dilute
media, may be brought out initially by a supplement of
nutrient broth or amino acids. 6) Target cells within
explants that respond specifically to auxins and cytokinins
should be selected and isolated.
Although callus may produce shoots (169), studies of
adventi tious bud formation in conifers tend to focus on
effects of cytokinins on epidermal and subepidermal cells
(11, 23, 24, 66). In Douglas-fir certain growth-regulator
combinations and high concentrations of nutrients tend to
produce callus from the spongy parenchyma. In spruce and
fir, urea and low calcium promote callus from epidermal
cells (Durzan, unpublished).
 279

In jack pine, the formation of callus from hypocotyl

and radicle explants correlated positively with the
nitrogenous composition of seeds, extent of imbibition and
germination, progress of metabolism associated with renewed
growth, and subcellular changes in ultrastructure (61).
Callus production was determined by the extent of earlier
contact between the germinating embryo and the haploid
nutritive gametophyte. When the dry, excised embryo was
placed on the agar medium, no more than 2% callus formed.
When the dry seed was cut in two and placed in water, each
half of the embryo grew, but no callus formed at the cut
surface. Work with Gingko (141, 142) and jack pine (97) has
shown that haploid tissues are a potent source of
growth-regulatory substances and nourishment for tissues in
vitro. In both cases, the haploid tissues are rich in
arginine and low in calcium.
The adenylate energy charge indicates whether synthetic
reactions are dominant over degradative ones (2). Jack pine
hypocotyls and radicles form little callus when the adenyl-
a te energy charge is be low 0.5 (61). When the charge is
greater than 0.5 much more callus is formed. Although the
adenylate energy status of cellular processes is controlled
by biosynthetic and degradative sequences, understanding of
this control is still incomplete (113 to 116, 168, 174).
Other factors, such as initial content of total soluble
protein and aminoacyl tRNA synthetase, were related nega-
tively and incompletely to callus dry weight (61). Electron
micrographs and cytochemical studies indicated that the
soluble proteins in the explant resided mainly in protein
bodies (62). When proteins were released from these bodies,
aminoacy 1 tRNA synthetase increased sharply as free amino
acids derived from protein started to accumulate. The
synthetase activity showed no direct relation to callus
formation. Even a high level of dry weight in the starting
tissue did not always lead to a greater final weight or
volume of callus (30). In the initial explant, dividing
280

cells were more responsive than nondividing cells to factors

in the medium leading to callus production.
At the callus surface the role of growth regulators is
obscured by the release of factors that condition the
medium, such as gases, free amino acids, glycoproteins,
lectins, and conjugates. If and how these correlate with
morphogenesis is not known. Morphogenesis sometimes ensues
with the removal of growth regulators or nitrogen sources
that were used to maintain the initial growth of cells (143,
145) .
3.3. Cell suspensions. Once a friable callus is estab-
lished, cell suspensions can be formed by transferring
callus to rotating or shaking culture systems containing
liquid medium of the same composition as in agar. The
callus fragments, cells and clumps become suspended in the
medium. The success in maintaining cultures for the inten-
ded purpose depends on the quality of cells, their origin
within the callus, the extent by which their metabolism can
be adjusted to the liquid medium, and a range of cultural
factors including inoculation density, subculture rate,
nutrient levels, growth regulator balance, aeration, and
culture vessel parameters (167, 168).
In cell suspensions of several plants, nitrogen is a
rapidly depleted ingredient of the nutrient medium (68,
174). Ammonium or reduced nitrogen is required for somatic
cell embryogenesis (77, 168).
3.3.1. Conifers. Suspension cultures have been estab-
lished with white spruce and jack pine (52, 58, 60) and
Pice a abies (29). In these instances, cells were cultured
in slowly rotating nippled flasks designed by Steward and
Shantz (144). At The Institute of Paper Chemistry (50) cell
suspensions of bther conifers have been established in
standard gyrotary Erlenmeyer flasks commonly used in many
laboratories. In another instance, conifer cells (Douglas-
fir) were grown in fermentors (Dougall, personal communica-
tion). These systems differ in several cultural parameters,
 281

which in turn can influence the way in which and rate at

which nitrogen is assimilated.
For jack pine cell suspensions, the overall growth in
dry weight tends to be near exponential (52). During the
first eight days, daughter cells separated to yield a finely
dispersed cell suspension, but from day 10 to day 18, new
daughter cells tended to adhere to form increasingly large
clumps. The amides and monosubstituted guanidines, includ-
ing arginine, accumulated in cells and in the medium (54).
The release of glutamine into the medium was higher in light
than in darkness and greater when clump size increased (55).
There are reports of other nitrogenous products being
released by cell suspension cultures, primarily arabino-
glycoproteins and lectins (6). Lectins have been defined
as sugar-binding proteins or glycoproteins of nonirnrnune
origin (92). Lectins are devoid of enzymatic activity
towards sugars to which they bind. This binding does not
require free glycosidic hydroxyl groups on the sugars.
Suspension cultures of Douglas-fir and loblolly pine cells
release lectins into media as part of the medium-clouding
phenomenon. Lectins may be used to bind nitrogen-fixing
bacteria to cells and tissues (10, 16, 17).
How do these nitrogenous compounds affect cells? When
L-glutamine or its analogues were added to isolated cells in
a medium containing sucrose and NAA, the rate of protoplas-
mic streaming increased (56). The same two substances
improved the streaming in cambial cells of pine (149). We
do not yet know if lectins have similar effects.
3.3.2. Acer. Studies of the nitrogen metabolism in
sycamore (Acer pseudoplatanus) have proceeded in four main
areas:
1) Nitrogen nutrition and assimilation,
2) The role of hydroxyproline-rich glycoproteins of
the cell wall,
3) Nucleic acid and protein metabolism, and
4) Intermediary nitrogen metabolism in cell suspen-
sions and synchronous cultures.
282

One of the first studies with cultured Acer cells investi-

gated the relationship between cellular respiration and
nitrogen metabolism. Givan and Collin (70) found that the
uptake of oxygen by cells was directly related to protein
synthesis and levels of intracellular nitrogen, and it was
more closely linked to cellular nitrogen metabolism than any
other parameters such as cell weight and rate of cell
division.
Comparisons of nitrate, ammonia, urea, and amino acid
mixtures as nitrogen sources have shown that nitrate is the
primary nitrogen source involved in cellular amino acid
biosynthesis in Acer suspension cultures (83, 84, 88, 174).
The regulation of nitrogen uptake has been studied by
monitoring the activities of various enzymes of nitrogen
assimilation (174). Acer cells in a steady state chemostat
with nitrate as the primary nitrogen source were fed gluta-
mate. The activities of nitrate reductase and urease were
depressed, whereas glutamate dehydrogenase and transaminase
levels increased in response to the available glutamate.
The intracellular glutamate and alanine levels became
elevated. The production of alanine was related to the
activity of glutamate-pyruvate transaminase.
The use of urea as a sole nitrogen source for Acer
cultures is still somewhat in question. Growth of cells in
urea is reported to be the same as or suboptimal to that in
medium containing nitrate (83, 88). Cells tended to exhibit
higher levels of intracellular glutamine, asparagine,
aspartate, glutamate, alanine, serine, and glycine accumu-
lated, although the total intracellular free amino acid pool
size remained constant (87).
The function of the accumulated free amino acids,
especially the amides, has yet to be demonstrated. The
amides should contribute nitrogen for the synthesis of
protein amino acids and purine and pyrimidine bases for
nucleic acids.
Supplementing a medium containing nitrate with casein
hydrolysate or a defined mixture of amino acids does not
 283

always result in optimal growth of cells. The fact that

lower cell densities are capable of better growth in condi-
tioned medium than in fresh medium led to attempts to grow
Acer cells in "reconstituted" conditioned medium (147). The
free amino acid content of conditioned medium was
determined, and these amino acids were then added to a
standard growth medium. The growth of the cells in this
artificially reconstituted medium improved but was not equal
to growth of cells grown in naturally conditioned medium.
It was suggested that a compound other than amino acids,
possibly volatile, released from cells was responsible for
the conditioned medium.
The carbohydrate metabolism of Acer cells may be
affected by nitrate reduction (84, 86). The reduced coen-
zymes NADH and NADPH are required for the nitrate reductase
and reductive amination of keto acids such as 0< -ketoglutar-
ate. The requirement for reduced coenzymes was postulated
to be met by increased carbon flow through the pentose
phosphate pathway (cf. Thorpe, this volume). In cells
supplied with nitrate, the pentose phosphate pathway was
stimulated, but the rate of glycolysis remained unchanged
(84) .
Interactions between nitrogen and carbohydrate metabo-
lism affect the synthesis and subsequent accumulation of
tannins in cultured Acer cells. Westcott and Henshaw (165)
reported that tannin synthesis is controlled by the initial
concentration of nitrogen and sucrose in the medium. High
levels of initial nitrogen, when replenished by further
addi tions after cultures started growing rapidly, delayed
the onset of tannin formation. By contrast, high levels of
sucrose in the medium led to greater tannin production.
With adequate medium nitrogen, the sucrose was thought to be
utilized in the synthesis of amino acids and proteins, but
when the nitrogen was depleted, the sucrose was diverted
into tannin production.
In cultured Acer cells, low levels of medium nitrogen
increased the activity of phenylalanine-ammonia lyase (PAL).
284

Cell lines with high PAL activity exhibited high levels of

phenolic synthesis (70). The same seems to hold for spruce
suspensions (27, 60).
The use of synchronous cultures has greatly contributed
to the study of nitrogen metabolism in Acer cells. The
technique has been especially useful in understanding the
cell cycle and the metabolism of nucleic acids, their
precursors and related enzymes. With a starvation and
regrowth treatment, cell cultures have exhibited synchrony
in cell division (90). Thymidine kinase activity and
thymidine incorporation peaked soon after cytokinesis (89,
90) suggesting that DNA synthesis occurred soon after
mitosis. The peak activity of aspartate transcarbamylase
was out of phase with DNA replication and was not related to
the rate of DNA synthesis as much as it was to the mainten-
ance of the nucleotide pool size. Thymidine utilization and
thymidine kinase acti vi ty have also been studied in normal
and bromodeoxyuridine-resistant cells (15).
The concentrations of nucleotides in cultured Acer
cells have been determined in lag, log, and stationary
growth phases (20). The analysis of spent medium indicated
a minimal leakage of nucleotides from the cells into the
surrounding medium. In a comparison of the routes and rates
of nucleotide synthesis, the free deoxyribonucleotide levels
were much lower than the intracellular ribonucleotides
(107). During the growth of cells, synthesis and levels of
RNA increased more than those of DNA. Increases in RNA and
DNA levels have been noted in the lag phase of growth (129),
but the subsequent cell divisions reduced the amount of DNA
per cell.
The metabolism of individual nucleotides have been
studied by use of radioactive substrates. Cox et al. (37),
using 14c-uridine, noted the uptake of uridine in all stages
of cell growth. Uridine was quickly incorporated into RNA
or used in the formation of phosphorylated compounds such as
uridine monophosphate. The labeled uridine was degraded to
 285

CO 2 , with a large proportion not reused or salvaged for RNA

synthesis.
The uptake and metabolism of exogenous adenine by Acer
cultures have been studied by Doree et al. (43,44) and
Doree (41, 42). The cells rapidly consumed adenine from the
medium, without evidence for intracellular accumulation.
Apparently the adenine is used immediately in the formation
of nucleotides. There also seems to be a rapid turnover of
adenylic nucleotides in cultured Acer cells.
The occurrence and role of hydroxyproline-rich glyco-
proteins in the cell wall of Acer has been reviewed by
Lamport (95). Lamport and Northcote (94) presented evidence
for the occurrence of a glycoprotein, later named
"extensin." The protein isolated from the cell wall
consisted of nearly 13% hydroxyproline unlike the
intracellular proteins, in which the proportion of
hydroxyproline was less than 1%.
Hydroxyproline-rich glycoproteins have been implicated
in determining the physical nature of the cell wall and in
effects of growth regulators on wall characteristics. One
hypothesis relates to the ability of auxins to increase the
plasticity of the primary cell wall to facilitate wall
extension. Upon investigation, the extending cell wall had
polysaccharide cross-linkages with labile bonds (95) which
were thought to play a role in auxin-induced cell wall
elongation.
The synthesis and transport of hydroxyproline-rich
proteins have been examined in cultured Acer cells (80, 94).
Proline is first incorporated into the protein and is then
hydroxylated to form hydroxyproline. This post-transla-
tional hydroxylation of proline uses atmospheric oxygen.
The synthesis of the protein is dependent on the
developmental state of the cultured cells. Autoradiographic
studies determined the distribution of exogenously supplied
14c-proline and revealed that the distribution of radioac-
tivity differs in growing and stationary-phase Acer cells.
1y growlng
I n ac t lve . ce 11 s, t h e 14C -pro I'lne was lncorporate
. d
286

into the cells and the nucleus (146). In older cells the
14C-label was localized in the thick cell wall suggesting
that the rate of protein synthesis varies with age of
cultured Acer cells. The hydroxyproline-rich glycoprotein
is synthesized in the cytoplasm and then transported to the
cell wall (39).
Another study involving Acer suspension cultures
indicated that "gas shock," or abrupt changes in gas concen-
tration, may affect cellular nitrogen metabolism (150).
Changes in the cellular uptake of the substrates leucine,
methionine, and adenine were noted after the culture flasks
were opened and the suspension cells were filtered. These
activi ties were presumed to change the composition of the
gases to which the cells were exposed and produce a gas
shock in the metabolism of the cells. This response to
culture vessel opening and sampling may become an important
consideration in the analysis of growth and biochemical
parameters of suspension cells.
The work with Acer and conifers (Appendix 1) contrib-
utes to our knowledge and to control of in vitro culture of
trees, and it has permitted a scale-up of cell suspension
cultures. These advances provide new opportunities for work
in applied areas of plant research that in the long run may
include vegetative propagation, secondary product formation
and even genetic engineering.
3.4. Morphogenesis. Visual or chemical markers are
needed to follow developmental patterns before we can obtain
a better understanding of the role of cellular and subcellu-
lar organization in cellular and tissue development (68, 85,
128). Subjects now being studied are the mechanisms of cell
division, the emergence of pattern and shape (11, 133), the
biophysical role of cell walls in pattern determination
(71), the evolving structure and function of subcellular
organelles, and the transformation of membrane systems, such
as rough endoplasmic reticulum (ER) to a smooth ER (93, 105,
109) . All these physiological activities in one way or
another involve growth regulators (64, 103, 134).
 287

FIGURE 7. Surface development of Douglas-fir cells derived

from cotyledonary explants during callus formation and
morphogenesis. A. Scanning electron micrograph showing the
emergence of a shoot (s) from a callus (C) on an agar plate
ugder the influence of (3-naphthoxyacetic acid (5 ppm) and
N -benzylaminopurine (0.1 ppm) on a MS medium (104). Note
the smooth surface of the shoot and the rough interior where
the surface has been fractured. Cells in the callus lack
288

cohesion and have a spongy appearance X80. B. When cells of

a suspension culture are placed on agar, a membrane (m)
forms at the air-cell interface and encases most cells.
Cell masses exposed to air develop a warty appearance of
pectin-like material (110)~ X480. C. Even while in suspen-
sion culture, some cells, shown here in cross-section
through light microscopy, develop a membrane that is easily
ruptured and sometimes floats to the surface of the culture
chamber~ X460. D. Electron micrograph of a cell in a
morphogenetic callus culture on agar showing the origin of
the membrane from the surface of the cell wall (W). Between
the membrane and cell wall is often a fluid, which when
analyzed is rich in t-aminobutyric acid~ X1600. E. Scanning
electron micrograph showing the contrast in cell surfaces at
the transition zone of shoot (s) formation and callus (c)
and the start of membrane (m) formation~ X250. F. Root from
adventitious bud has a membranous cover (m) X120. Figures A
to 0, courtesy of H. Kaustinen, and Figures E and F, John
Litvay. Observations of D. J. Durzan.

A significant transformation of the rough ER is now

thought to occur under the influence of auxins and cytoki-
nins and after changes in the nucleus. The rough ER is
replaced by a smooth ER (no polysomes) which, in turn,
produces somehow a membranous coating outside of cells that
eventually "packages" cells showing incipient morphogenesis
(Fig. 7). These outer membranes may be polyesters (93) or
proteinaceous (98).
In white spruce cell suspensions grown at high levels
of Ca(N0 3 )2' another prominent product, tannin, arises via
the aromatic family of amino acids and produced by the rough
ER (27). Under fluctuating and stressful environmental
condi tions inhibitory to morphogenesis, tannin bodies seem
to originate in the cytoplasm by fusion of vesicles emerging
from the rough ER. These bodies produce tannin, which
accumulates on the surface of the cells and in vacuoles. In
the latter case, the vacuoles are often filled with the
tannins through the proliferation of vesicular or filamen-
tous membranous whorls.
Sphaeroblast-like growth was observed when the internal
secretion of tannin was reduced by culturing spruce cells
under constant optimal environmental conditions (60). In
cells of American elm that eventually regenerated into
 289

plantlets, tannins accumulated at the plasmalemma, and outer

cell wall, where membranes were observed (Chafe and Durzan,
unpublished) .
3.4.1. Nitrogen metabolism of natural embryos. Workers
attempting experimental morphogenesis should benefit from
knowledge gained from natural embryogenesis (22, 40, 145).
Unfortunately, little is known about nitrogen metabolism in
natural embryo development of most forest trees. Presumably
metabolic patterns similar to those found in embryos occur
after the rejuvenation or invigoration of cells. The
developing zygote of conifers shows initially considerable
dedifferentiation of cytoplasm. The dense staining of
cytoplasm suggests an elevated heterotrophic nutrition,
which, in turn, suggests that the dominant reactions during
dedifferentiation are initially degradative.
The enriched pool of nitrogenous compounds contributes
to the formation of new nucleic acids and proteins. The
mul tinuclear stage in early conifer embryogenesis suggests
that the sequence of information flow from DNA, and RNA to
protein and cellulose is somehow temporarily blocked, thus
preventing cell walls from forming. The limited evidence
with developing Douglas-fir seeds indicates that early
stages are characterized by high levels of free amides and
proline. The carbon flow in the young embryo appears to be
determined by the fat and lipid reserves of the seed as
mediated by peroxisomes and glyoxysomes (153).
As the proembryo develops cotyledons, the amides and
especially glutamine buildup in the free amino acid pool.
Thereafter, much of the nitrogen metabolism seems to be
diverted to protein and nucleic acid synthesis for forming
reserve proteins in protein bodies and for replicating
nucleic acids as cells divide. This is paralleled by an
increase in free and bound arginine. A similar free amino
acid pattern occurs in buds flushing in late spring (46) or
in vitro (28).
Research in literature on the nitrogen metabolism of
germinating conifer embryos is scattered. Attention has
290

been paid to high levels of arginine (101), the ornithine or

urea cycle (106), the arginine-rich storage proteins (119),
and the high levels of DNA in the nucleus of endosperm and
embryo tissues (117). More recently, the nucleotide pool
and biology of chromatin has received more interest (112,
114, 115, 116).
The fact that experimental morphogenesis can now be
achieved in cell suspensions of woody species opens new
possibilities for studying the hereditary and developmental
mechanics of cells and tissues undergoing morphogenesis.
The trend in several laboratories with cell suspensions is
to attempt to mimic in vitro the nutritional and biochemical
events that typify true embryos in situ.
Whole embryos of red pine (Pinus resinosa) excised at
various stages of development and cultivated in vitro showed
good potential for growth and differentiation (5). The
nutrient medium contributing to the new developmental
expressions of the isolated embryos consisted of a simple
inorganic medium lacking nitrogen (KC1, caso 4 , MgS0 4 , MgP0 4 )
but supplemented by an aqueous extract from the Ginkgo
gametophyte known to be rich in arginine. Banerjee and
Radforth (5) found that the polarity already imposed on the
post-zygotic mass could be reoriented. The Ginkgo extracts
facilitated normal embryonic development when the embryo was
already elongated and had cotyledons but this was not the
case with embryos at the globular stage. When embryos with
delineated apical meristems were excised and cultured, they
elongated and formed numerous secondary embryos.
The formation of embryos from excised suspensors and
from the suspensors of intact embryos grown in vitro was
explained as arising through a loss of inhibitory influences
(5) . Inhibi tion was a function of cell position on the
embryonic axis and presumably was determined by metabolic
gradients arising from the gametophyte. The latter no doubt
involve inhibitory nitrogenous compounds, possibly those
derived from arginine such as the highly inhibitory monosub-
stituted guanidines and amines (54).
 291

Thomas (151, 152), studying excised embryos of Pinus

silvestris, found that the removal of vitamins and high
levels of sucrose inhibited embryonic development. However,
of severa.l media tried, the one described by Halperin (77)
(even with vitamins and lower sugar levels) did not encour-
age growth until after the initiation of cotyledons; coty-
ledons supply organic nitrogen for the development of
embryonic axis (12, 19, 62). This points to the need for an
organic nitrogenous supplement at the earlier stages of
development and to the need to control antagonism between
nitrate and reduced nitrogen in typically nitrate-rich
media.
3.4.2. Somatic embryogenesis. In the wild carrot,
reduced nitrogen and the removal of 2, 4-D, are needed to
induce free cells and clumps to form somatic embryos (145,
166) If embryos of forest trees could be induced in
suspension at the same rate as carrot embryos (77), then, in
only 100 liters of medium, 73 million plantlets could be
produced and raised in containers for field planting (24).
The problem is how to induce somatic embryogenesis. At
this time there are three examples of putative somatic
embryogenesis in forest trees. Wherever possible these are
evaluated from the viewpoint of nitrogen metabolism.
3.4.2.1. Sweetgum (Liguidambar styraciflua). Sommer and
Brown (136) germinated seeds of sweetgum, which were trans-
planted to an agar medium of major salts and vitamins that
contained Ca (N0 3 ) and KN0 3 but no ammonium N. Iron was
amine-bound and provided as Fe-EDTA. Vitamins containing
nitrogen included thiamine, nicotinic acid, and pyridoxine.
After one month of growth on the medium without growth
regulators, the hypocotyl segments were placed on a second
medium containing ammonium nitrate and 25 combinations of
NAA and BAP. After two months, bud and root differentiation
occurred on media with high and low combinations of BAP/NAA,
respectively. When cultures were transferred to media of
the same composition but lacking the above growth regulators
292

and when the nutrient medium was replenished weekly, embryos

were developed after four months.
Resul ts of trials indicated no clear evidence for a
specific' combination of BAP/NAA for the induction of embryo-
genesis (136). Sommer and Brown felt that the depletion or
absence of some unidentified constituent in the medium was
important in releasing the totipotency of the sweetgum
cells. If this is true, then some nitrogenous compound
should be a good candidate.
Although they did not emphasize it in their initial
paper, Sommer and Brown changed the form of nitrogen avail-
able to cells by switching from medium I to medium II. The
nitrogen was initially in the oxidized state as nitrates but
was then switched to an enriched, balanced form of nitrogen
(NH 4 N0 3 , KN0 3 , and Ca(N03)24H20). Ammonium is a requisite
for carrot embryogenesis (166) and it is also readily
depleted from the medium so the switch in nitrogen may have
contributed to the induction of embryogenesis in sweetgum.
The entry of ammonium and nitrate nitrogen into organic
combination depends on asparagine and glutamine as ports of
entry (Figs. 2, 3). The question remains how the balance of
C4 and C5 amides (asparagine and glutamine) controls embry-
onic development in the presence or absence of growth
regulators. One possibility is that amides contribute to
purine synthesis in the de novo (31) pathways associated
with the production of endogenous cytokinins and nucleotides
(59). Where nucleic acids are degraded, the amides can
receive the nitrogen released from the bases, and where the
bases are salvaged for renewed nucleic acid synthesis, the
amides can again contribute nitrogen for base modifications
(uracil to cytosine and methyl cytosine). Another possibil-
ity may relate to the switches in N metabolism associated
wi th the flow of nitrogen from amides to arginine, or a
similar storage product, activated or inactivated by synthe-
tic growth regulators (e.g., Table 2) or their conjugates
(Table 1).
 293

3.4.2.2. Douglas-fir and loblolly pine. For cell

suspensions of conifers, an example of the early stages of
putative somatic embryogenesis is that for Douglas-fir and
loblolly pine at The Institute of Paper Chemistry (1977-
1981) . This work is an extension of earlier studies of
Durzan and Steward (58), with supplements of coconut milk
and extracts of female gametophytes.
Cell suspensions from the buds and shoot apices of
3-to-4-year-old potted and mature trees on defined media
have yielded putative somatic proembryos but only to the
partly polarized stage (50) . Subsequent development resem-
bled, sphaeroblasts, and several species (jack pine, white
spruce, Douglas-fir) even rooted (Figs. 8 and 9). Tempera-
tures were from 20 to 23C, and the constant light did not
exceed 3 uW cm 2 in the blue and red visible spectrum.

FIGURE 8. Elongated somatic proembryos derived from coty-

ledonary cells (160 to 280 dial of Douglas-fir after 120
days in liquid suspension culture tend to develop internally
like sphaeroblasts; X150.
294

FIGURE 9. Longitudinal sections of sphaeroblasts of

Douglas-fir from same cell line in Fig. 8. The sections
illustrate the organized growth patterns of internal cells.
In these studies, the root-like organs developed
precociously and the morphogenesis tended to be more
characteristic of organogenesis rather than embryogenesis
X150 (Durzan, unpublished observations).
Concerning the interpretation of N-metabolism and
nutrition in these cultures, the initial explant has gen-
erally been exposed to high levels of NH 4 N0 3 (800 ppm) and
growth regulators (2.5 to 5.0 NOAA and 0.1 BAP) to favor
callus production. NOAA appears to inhibit morphogenesis in
the callus. It tends to produce callus from cells in the
parenchyma rather than from the epidermal cells, unless less
calcium and more reduced nitrogen are used. When calli are
placed in suspension culture, NOAA in the medium is replaced
by NAA and the nitrogen and salt levels are lowered by a
half to a quarter. This facilitates the production of a
cell suspension. After 5 days, the cells are screened,
collected (60-to-280;U diameter fraction) and inoculated
 295

into a specially formulated medium low in calcium, lacking

ni trate and synthetic growth regulators, and containing a
wide range of free amino acids and amides. The embryonic
structures will form with ammonium or urea as the sole
source of nitrogen. More recently, the commonly used
medium, such as that of Murashige and Skoog (104) but
diluted, has proven effective with low levels of cytokinin
(0.1 ppm) and auxin (0.01 ppm).
The induction of organized growth involves low levels
of reduced nitrogen. This leads to internal cell division,
resulting in reduction in cell size in some but not all cell
lines. Internal divisions are fostered by the addition of a
free amino acid supplement. Free amino acids also contri-
bute to embryogenesis in free pollen cultures of the potato
(163)
Arginine is a commonly found amino acid in conifer
seeds and leads to the formation of ornithine and polyamines
via ornithine decarboxylase. ~-Methylornithine added to the
medium as an inhibitor of ornithine decarboxylase does not
inhibit the formation of globular embryoids.
The proteolysis that occurs during cellular dedifferen-
tiation with cells in impoverished media provides hetero-
trophic nutrition equivalent to the amino acid supplement.
After 3 to 4 weeks, cells in suspension grow into small
globular nodules. After one to two months, the globular
structures begin to polarize. At this stage they are
transferred to fresh liquid medium to encourage continued
development.
Polarity is expressed in terms of an elongated morphol-
ogy, the development of a central core of cambial-like
cells, linear array of cells constituting the epidermis, and
the emergence of meristematic cells at the poles of the
structure (Figs. 8, 9). Very often precocious vascular
development, typical of sphaeroblasts, occurs. This is
common in cell lines from more mature trees and seems to
arise from the response to growth regulators and sugar in
296

the medium: the older cell lines revert to a more mature

expression than do cells from juvenile sources.
In a third example, Weyerhaeuser was granted a u.s.
patent (#4,217,730, August 19, 1980) on somatic embryogen-
esis and organogenesis (1). An embryoid was defined as "an
asexually reproduced bipolar group of organized cells having
defined meristemic centers that can ultimately produce roots
and foliage, together with an appropriate vascular system
for internal transport of nutrients and waste products. It
differs from seed embryo primarily in the asexual method of
development which gives genetic identity to the tissue
donor."
The medium for embryoid formation contains errors and
was a full-strength Murashige-Skoog (104) formulation
modified to include higher levels of myoinositol (250 ppm)
and thiamine (2.5 ppm) (EI-Nil, personal communication).
The Weyerhaeuser study differed from the two cases above in
that no overt change in nitrogen levels were attempted.
3.4.3. Organogenesis. So far, organogenesis has been
studied with juvenile rather than mature trees because the
process is more successful with juvenile trees (85). The
controlled formation of new organs on explants of conifers
provides a means for studying developmental problems from
the viewpoint of nitrogen metabolism.
In Douglas-fir hypocotyls, organogenesis occurs usually
in three steps. The first involves the action of synthetic
growth regulators on explants. The second is the removal of
this stimulus. The third is stimulation of the meristem-
oids or sphaeroblasts to develop into adventitious buds and
roots. Recently, however, with Douglas-fir cotyledons and
with 10 ppm NAA on Cheng's medium (32), roots were initiated
in darkness while buds were initiated in yellow and red
light (560 nm and 660 nm) at high intensity (Tran Thanh Van
and Yilmaz, personal communication). Earlier, in white
spruce hypocotyls lacking an apical meristem, the shoot-
producing stimulus was more directly related to the synthe-
tic adenine derivative N6 -benzylaminopurine, and rooting was
 297

a function of polarity and high levels of c:J,. -naphthalene-

acetic acid (24).
From the viewpoint of nitrogen metabolism, BAP or NAA
influences the behavior of chromatin DNA (112) and forms
conjugates either with other nitrogenous products, such as
the amino acids, or with glucose (Table 1). The signifi-
cance of these conjugates remains to be demonstrated. Some
speculation exists that conjugates serve as internal
reserves for growth regulators (72). We have seen (Table 2)
that the auxins and cytokinins also change the pattern of
amino acid composition of cells.
Cytokinins, being derivatives of adenine, commonly
degrade to urea (59) and contribute to the synthesis of new
nucleic acids and to the establishment of new centers of
growth activity (99, 102) Although they also inhibit
respiratory kinases, they do not affect the activity of
glutamine synthetase (158) . The cytokinin dependent induc-
tion of certain enzymes, such as cyclic nucleotide
phosphodiesterase, can be inhibited by a calmodulin
inhibitor, trifluoperazine, even in the presence of calcium
(63). None of this explains how organogenesis starts or how
it leads to the launching of the development of meristemoids
or sphaeroblasts.
An extension of organogenesis into protein metabolism
was described by Cheng (33) and Hasegawa et al. (79). In
Douglas-fir, proteins of low molecular weight (16,000 to
20,000 daltons) appeared as early as two days after the
inductive treatment for adventitious buds. The authors felt
that this was the earliest biochemical event associated with
morphogenesis that they could detect. However, during the
induction of buds in cotyledons or comparable tissues, there
was inevitably some loss of cellular structure in cells
adjacent to centers of morphogenesis. Since this loss may
contribute breakdown products and atypical new proteins,
further investigations should take this concern into
account. Nevertheless, the postulate of the induction of a
specific protein associated with organogenesis, or with any
298

other aspect of development, is important for our

understanding of organogenesis.
Morphological polarity has been explained by polar
auxin transport (164). To induce organogenesis in conifers
requires much higher levels of auxin than those normally
found endogenously (1 to 10 nanomoles). It has been sugges-
ted (105) that high exogenous auxin acts on membranes of the
cell. Auxin also contributes to increased enzymic activity
rather than to enzyme synthesis itself (162).
Added auxin lowers the pH of the medium through the
pumping of H+ ions out of the cell (162). The low pH
activates enzymes already in the wall and promotes the
uptake of chloride. Chloride and other monovalent anions
strongly activate the glutamine-dependent synthesis of
asparagine and the hydrolysis of glutamine (123).
Subsequent reactions of auxins seem to promote the
secretion of wall-modifying enzymes and wall precursors (71)
or involve the formation of auxin conjugates (4, 78, Table
1). Conjugates with nitrogenous compounds involve the
dicarboxylic amino acid aspartate, and the bond involved in
the conjugation is an amide or peptide bond. Some conju-
gates regulate growth (78), and 2,4-D derivatives have been
implicated in the control of carrot embryogenesis (100).
Peroxidase rapidly oxidizes IAA but not the IAA conjugates
of amino acids (35).
Most concepts based on the addition of exogenous auxins
are believed to involve indoleacetic acid oxidase, a peroxi-
dase-type enzyme that uses H20 2 to degrade IAA to indolenine
hydroperoxide. Presumably degradation is far more rapid
than is the de novo synthesis of auxin from tryptophan via
indoleacetaldehyde (127). In developing cellular systems,
nitrogen metabolism soon becomes integrated with other
metabolic cycles, such as the metabolism of phenolic com-
pounds and their effect on indoleacetic acid oxidase.
Flavonoids with a catechol B-ring inhibit plant oxidase
activity and thus are believed to spare IAA: they free it
to maintain hormonal balance and plant growth. By contrast,
 299

if the flavonoid has one hydroxyl moiety on the B-ring, the

relative enzyme activity may be augmented to reduce IAA
levels and inhibit growth (139). Hydroxycinnamic acids and
3,4-dihydroxyphenols such as caffeine (a purine) have
similar effects. Further, phenolics may affect the pathway
of auxin biosynthesis from anthranilic acid and tryptophan.
Flavonoids may also affect the polar transport of auxins
(140) .
The mechanism of action of the auxin protectors in
Douglas-fir is supported by observations that the lag phase
in the oxidase reaction may be eliminated by adding perox-
ide. Presumably the auxin protectors bind to the enzyme.
Upon standing, the auxin protector-IAA oxidase preparations
tend to turn brown. The brown substance released from the
enzyme gives a positive reaction of o-diphenols. In light
of these and other reasons (26), the reports of beneficial
effects of adding phenolics (e.g., homogentisic acid, DOPA)
take on added significance, particularly in rooting efforts
(135) .
The above deals with organogenesis at the molecular
level and in already organized tissues. In callus, when
organized meristemoids emerge, strongly adhering daughter
cells are clearly evident (Fig. 7). These cells seem to be
linked by acidic polysaccharides and polyester membranes
which serve as an intercellular matrix and binding material.
Apparently the polysaccharides possess a cation retention
and exchange capacity which may make them reservoirs of ions
and nitrogenous compounds at the cell surface (120). As
cells continue to divide, the meristemoid produces lineages
of cells or meromeres (74, 155), and these are enclosed, at
least under in vitro conditions, by a yet-uncharacterized
membrane (Fig. 7).
In organizing cells in suspension cultures, the
tendency is to form the membrane at liquid-air interfaces so
as to encase and bathe the cells in a liquid. Preliminary
analyses of this membrane showed that it contained bound
sugars and amino acids. Fluid under the membrane (Fig. 7D)
300

was rich in y-aminobutyric acid and possibly CO 2 when the

acid was formed by decarboxylation of glutamic acid. In
this context, some seed coats when moistened release
phenolic compounds that combine with oxygen and release CO 2
(36). This points to the need to consider gaseous exchanges
carefully, especially when growth and development are
activated (75).
Ultimately the membrane over cells may lead to the
formation of a smooth epidermis containing cutin, suberin,
and phenolic substances (93). It seems that the surface
membrane is but one of several indications of morphogenesis
at the cellular level.
Further integration of biochemistry and physiology with
morphogenesis will be needed.

4. OUTLOOK
Since nitrogen is often a factor limiting growth in
situ ana in vitro, all aspects of vegetative propagation and
silviculture could benefit from the understanding and
application of nitrogen. Apart from the use of slow-release
fertilizers, the opportunities and technologies to do this
economically are not immediately obvious (25). However,
recent aevelopments, particularly in the provision of
controlled rates of active substances and use of monoclonal
antibodies, may help remove these shortcomings (159).
One opportunity lies with the introduction and control
of symbiotic nitrogen fixation (10, 16, 17). It may be
possibl e to genetically engineer plant cells, as has been
done with microorganisms. Although this goal may be real-
ized someday, the value of this technology to forestry
remains to be proven. Using lectins, we may be able to bind
nitrogen-fixing organisms not only to the rhizosphere but
also to the aerial parts (e.g., needles) of trees. It may
also be possible to make improvements in the cloning cycle
(50, 54, 71), such as the formation of artificial seeds where
the somatic embryo is coated to form a pellet; this would
proviae ease of handling, protection, and the slow release
of nutrients (103). Also possible may be the use of
 301

haploid nuclei and protoplast technology to recreate the

mul tinuclear stage of the zygote and see how this contri-
butes to nitrogen metabolism during subsequent growth and
development.
Our understanding of the nitrogen metabolism of trees
compared with that of agricultural plants is deficient.
Only recently have we been able to reduce trees to their
cells and reconstitute meristems and young trees
(Liquidambar) for the field. These systems could contribute
to a wider range of baseline information needed to properly
interpret physiological states through nitrogen metabolism
and development. This will include testing of the recent
advances coming from animal studies such as the role of
calmodulin in calcium-mediated reactions of nitrogen
metabolism. Calmodulin action includes effects on protein
kinases, cyclic AMP, and NHCP and the control of gene and
enzyme expression. We can anticipate that a wider range of
enzymes and physiologically active substances will be
encountered such as DNA repair enzymes, peptides, and
phenolic conjugates.
One of the great deficiencies in morphogenesis and
nitrogen metabolism is that most studies have not been
linked to heredity. The expectation is that as industry
defines its desirable gains, these will be redefined in
molecular terms and at the level of chromatin. The hope is
that proven traits will be mass-produced for the industrial
forest independent of climate and season. Tree cells may
someday be exploited like microorganisms, and new characters
may be engineered or chemical products mass-produced. The
incentive is no longer merely curiosity. For the forest
biologist, numerous challenging problems will remain. These
are based on the organization of tissues in the tree over
all phases of the life cycle and in relation of trees to the
nitrogen cycle in nature (148).
302

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 313

Appendix 1. Annotated and chronological summary of selected

references on the nitrogen metabolism of forest
trees in culture. A. Gymnosperms B. Angiosperms

A. Gymnosperms

Year Author & Reference Species & Remarks

1924 SCHMIDT A. Bot . Arch. Pinus, Picea, Biota: effect

5:260-282 "()f"Nsources on chloro-
phyll formation in excised
embryos

1936 RADFORTH NW. Trans. Ginkgo proembryos in

R. Can. Inst. vitro, calcium nitrate and
Toronto . 21:87-94 yeast extract as nitrogen
sources to sustain devel-
opment

1955 REINERT J Naturwiss- Picea glauca, role of cop-

enschaften 42:18-19 per containing enzymes and
tyrosine in browning

1956 REINERT J & WHITE PRo Picea glauca: normal and

Physiol. Plant. 9: tumor tissue tyrosine,
117-189 phenylalanine, glutamine,
glutamate, asparagine,
aspartate and Vitamin B12
promote growth

1956 RISSER PG & WHITE PR o Picea glauca, tumors,

Physiol. Plant . 17: glutamine as a suitable N
620-635 source

1957 RESTOOL DF. Diss. Abstr. Sequoia, cysteine and B-

17:734-735 vitamin effects on bud
formation and callus
growth

1958 BARNES RL & NAYLOR AW. Pinus clausa: callus, 14 C_

Bot. Gaz. 120:63-66 glutamic acid metabolism

1959 BARNES RL & NAYLOR AW. Pinus serotina, Pinus

Bot. Gaz. 121:63-69 CIaUSa: ornithine cycle in
roots and callus

1959 BARNES RL & NAYLOR AW. Pinus serotina: root cul-

For. Sci. 5:158-168 ture, glycine and argin-
ine effect on growth, me-
tabolism of glutamic acid
and aspartic acid
314

Year Author & Reference Species & Remarks

1959 BARNES RL & NAYLOR AW. Pinus serotina: root cul-

Physiol. Plant. 12: ture, nitrate urea and 7
82-89 amino acids tested as ni-
trogen sources. Nitrate,
t-aminobutyric acid and
citrulline best

1959 REINERT J & Picea glauca in contrast

SCHRAUDOLF H. Planta to crown gall tumorous
53 : 18-24 cells of spruce, can be
grown only in presence of
IAA

1960 TULECKE W. Plant Ginko biloba: callus,

Physiol . 35:19-24 arginine required for
growth

1961 BARNES RL. Bot. Gaz . Pinus elliotti, Pinus

123 : 141-143 paIUStris, l~nusCIaUsa:
Adenine -8- C catabolism
in embryos and callus
cultures

1961 BARNES RL & NAYLOR AW. Pinus palustris, Pinus

For . Sci. 7:130-135 elliotti: excised embryos,
metabolism of ornithine

1961 DE TOROK D & Picea glauca: asparagine

THIMANN KV. Physiol. as nitrogen source for
Plant. 9:177-189 normal and tumor tissues
in culture

1961 STEINHART CE, Picea abies: callus,

STANDIFER LC, SKOOG nutrient requirements,
F. Am . J . Bot. 48: arginine supplement
465-472

1962 BARNES RL. Nature Pinus clausa, Pinus

(Lond.) 193: 781 eIIIOtti, Pinus-5erotina :
'(-guanidinobutyri~ acid
production from C-
arginine by tissues

1962 BARNES RL. Plant Pinus taedt4 Pinus

Physiol. 37:323-326 serotina: C-glutamic
acid metabolism in
isolated roots
 315

Year Autho r & Refere nce Speci es & Remar ks

1962 BARNES RL & NAYLOR AW. Pinus palus tris,. Pinus

Plant Physi ol. 37: ellio tti, Pinus clausa :
171-17 5 callu s, f-> -alani ne pro-
ductio n, aspar tic acid
and uraci l metab olism

1962 HIGUCHI T. Can. J. Pinus strobu s callus

Bioche m. Physi ol. l~gnin synth esis using
40:31- 34 C-phe nylala nine

1962 TULECKE W, WEINSTEIN LH, Ginko biloba : callu s, free

RUTNER A, LAURENCOT HJ. amino acids of pollen tis-
Contr ib. Boyce Thomp son sue cultu re, ungerm inated
Inst. 21:291 -302 pollen , leaves and nucel -
lus compa red

1963 KLEIN RM Physi ol. Plant . Ginkgo an argini ne and

16:73 -81 auxin requi ring cells of
pollen and effec ts of dif-
feren t wavel engths of
light on growth

1963 STRAUS J & CAMPBELL WA. Cupre ssus callu s, enzym es

Life Sci. 2:50-6 2 releas ed into the medium
under influe nce of caC1 2

1964 BARNOUD F et al. C. R. f;quo ia callu s, fate of

Acad. Sci. Paris 259: C-phe nylala nine
4339-4 341

1964 ENGVILD KC. Physi ol. Pinus jeffre yi: excise d

Plant . 17:866 -874 embryo cultu re, urea and
recon stitut ed megag ameto-
phyte amino acids as ni-
trogen source s

1964 RISSER PG & WHITE PRo Picea glauc a: callu s,

Physi ol. Plant . 17: amino acids as nitrog en
620-63 5 sourc es, adenin e,
~-alanine empha sis on
'tyros ine, glutam ine and
cystin e

1964 WHITE PR & NORTON S. Picea glauc a: nitrog en

Plant Physi ol. source s other than
39: suppl . 1 XIV glutam ine

1965 CONSTABEL F & KIRSTEN G. Junip erus commu nis:

Ber. Dtsch . Bot. Ges. chang es in amino acid com-
78:38- 43 positi on and pH in cultu re
medium
316

Year Author & Reference Species & Remarks

1965 CONSTABEL F, KIRSTEN Juniperus communis: free

G & STEINER M. Ber. amino acids of cells in
Dtsch. Bot. Ges. culture, decreased argin-
78:348-353 ine, citrulline and histi-
dine in auxin-heterotropic
cells

1965 LAMPORT DTA Adv. Bot. Ginkgo callus (n and 2n)

Re s 2: 151- 218 amino acid composition of
cell wall proteins

1965 NURSTOG K. Am. J. Zamia integrifolia nitro-

Bot. 52: 993-999 genous supplements used to
induce apogamy in mega-
gametophytes

1965 TULECKE W & RUTNER A. Ginko biloba: suspensions,

In: White PR & Grove amino acid composition of
AR (eds) Proc. cells and medium
Int. Conf. Plant
Tissue Culture,
McCutchan Publ.,
pp. 103-116

1966 HIGUCHI T & BARNOUD FJ. Sequoia and' Ginkgo callus,

Jpn. Wood Res. Soc. phenYi!anine deaminase and
12:36-43 fate C-phenylalanine

1966 WHITE PR & GILBEY SN. Picea glauca: nitrogen

Physiol. Plant. 19: sources
177-186

1967 DAVID A. C.R. Acad. Pinus pinaster: free amino

Sci. Paris 264:919- acids in cultured cells in
921 darkness

1967 DAVID A. C.R. Acad. Pinus pinaster: free amino

Sci. Paris 265:1602- acid changes during callus
1605 initiation

1967 TULECKE W. Am. J. Ginkgo and Taxus: d -amino-

Bot. 54:797 levulinic acid and
synthesis

1967 TULECKE W Phytomor- Ginkgo biloba: free amino

phology 17:381-386 acid composition of callus
and explant source

1968 BROWN CL & LAWRENCE RH. Pinus palustris: callus,

For. Sci. 14:62-64 nitrogen sources of cul-
ture medium, asparagine
 317

Year Author & Reference Species & Remarks

1969 HARVEY AE & GRASHAM JL. 12 species of conifers:

Can. J. Bot. 47:547-549 callus, medium compos-
ition (Abies, Larix,
Picea, Pinus, PSeUdotsuga,
Thuja, etc.)

1970 ROGOZINSKA JH. Acta Pinus sylvestris: callus,

Soc. Bot. Pol. 39: nutritional requirements
151-160 (arginine, tryptophan,
glutamine and tyrosine)

1971 DAVID A. C.R. Acad. Pinus pinaster: medium

Sci. Paris 273:2236- composition for callus
2238 initiation

1972 DURZAN D & CHALUPA V. Pinus banksiana: changes

Proc. Can. Soc. Plant in amino acids during
Physiol. 12:22 callus and seedling growth

1972 KONAR RN. Final Report Pinus gerardiana: gluta-

USDA Project PL480 mine and arginine promote
growth of cultures, 21
amino acids tested as
nitrogen sources

1973 CHALUPA V & DURZAN DJ. Picea glauca, Picea

Can. J. For. Res. mariana, Picea abies,
3:196-208 Abies balsamea,-----
PSeUdotsuga menziesii:
amino acid composition in
buds similar to buds under
field conditions and con-
trollable by auxins and
cytokinins

1973 DURZAN DJ, CHAFE SC & Picea glauca: suspensions,

LOPUSHANSKI SM. Planta effect of environmental
113:241-249 changes on phenylalanine
levels and organized
growth

1974 GREENWOOD MS, HARLOW AC Pinus lambertiana: excised

& HODGSON HD. Physiol. embryo segments, formation
Plant . 32:198-202 of NAA-aspartic acid con-
jugates

1974 KONAR RN. Physiol. Pinus gerardiana: callus,

Plant. 32:193-197 medium composition, gluta-
mine in medium
318

Year Author & Reference Species & Remarks

1974 MOMOT TS et al. Izv. Picea abies: callus, amino

Akad. Nau~ SSSR. acid biosynthesis from
Ser. Biol. 5:666-671 sucrose

1975 CAMPBELL RA & OURZAN OJ . Picea glauca: callus,

Can. J. Bot. 53:1652- importance and use of
1657 auxin/cytokinin ratios to
form new meristems

1975 MOMOT TS. Izv. Vuz. Picea abies: root culture,

Lesn. Zh. USSR. 18: amino acid synthesis and
36-38 metabolism

1976 OURZAN OJ & BOURGON G. Pinus banksiana: callus

Can. J. Bot. 54:507- and suspensions, effect of
517 L-glutamine and its
analogs on subcellular
activities

1976 OURZAN OJ & CHALUPA V. Pinus banksiana: callus,

Can. J. Bot. 54:468- changes in the amino acid
483 composition during growth
compared with seedlings

1976 OURZAN OJ & CHALUPA V. Pinus banksiana: callus,

Can. J. Bot. 54:453- changes in arginine and
495 monosubstituted guanidines
during growth compared
with seedlings

1976 OURZAN OJ & CHALUPA V. Pinus banksiana: suspen-

Can. J. Bot. 54:496- sions, effect of light
506 and darkness on free amino
acid composition

1976 OURZAN OJ, CHALUPA V Pinus banksiana: medium

& MIA AJ. Can. J. Bot. composition, relationship
54:437-445 of callus formation and
biochemical state of
explant source

1977 BONGA JM. In Vitro 13: Abies balsamea: effect of

41-48 nitrogen compounds

1977 CHENG T-Y. Plant Sci. Pseudotsuga menziesii:

Lett. 9:179-187 callus, effect of growth
regulators on morpho-
genesis
 319

Year Author & Reference Species & Remarks

1979 CHENG T-Y. In: WR SHARP, Pseudotsuga menziesii:

PO LARSEN, EF PADDOCK callus, gene products
& V RAGHAVAN (eds.) affecting protein syn-
Plant cell and tissue thesis and morphogenesis
culture, Ohio State
Univ. Press, pp. 493-
508

1979 HASEGAWA PM, YASUDA T Pseudotsuga menziesii:

& CHENG T-Y. Physiol. callus and cotyledon
Plant. 46:211-217 culture, proteins assoc-
iated with cultures cap-
able of adventitious bud
formation

1979 JOHNSON MA & CARLSON JA. Pseudotsuga menziesii:

Biochem. Physiol. callus, indoleacetic acid
Pflanz. (BPP) 174: 115-127 oxidase

1979 KIRBY EG & CHENG T-Y. Pseudotsuga menziesii:

Plant Sci. Lett. protoplasts, effect of
14:145-154 glutamine on cell division

1979 MUREN RC, CHING TM & Pseudotsuga menziesii: In

CHING KK. Physiol. vitro pollen germination,
Plant. 46:287-292 metabolic study

1979 RAMAWAT KG & ARYA HC. Ephedra gerardiana, ~.

Phytomorphblogy 29: foliata: callus, effect of
16-25 nitrogen sources on growth
and protein synthesis

1979 RIOV J, COOPER R & Pinus pinea, Pinus

GOTTLIEB HE. Physiol. halepensis: formation of
Plant. 46:133-138 aspartic acid conjugates
of NAA and IAA by tissue
segments

1979 VANDIVER VV & FITES RC. Pinus taeda: callus,

Plant Physiol. 64: thymidylate synthase
668-670

1980 DUHOUX E & THI AT. Juniperus communis: pollen

Physiol. Plant. 50: culture, influence of
6-10 amino acids on growth

1980 KIRBY EG & FRANK PS. Pseudotsuga menziesii:

Plant Physiol. 65: suspensions glutamic acid,
supplement 37 glutamine and allantoin as
nitrogen sources
320

Year Author & Reference Species & Remarks

1980 LAU YL, SHELD HW & Pinus elliottii: callus,

COWLES JR. Physiol. phenylalanine ammonia-
Plant. 49:299-303 lyase activity

B. Angiosperms

1951 JACQUIOT C. C.R. Acad. Ulmus capestris: effect of

Sci. Paris 233:815-817 inositol and adenine on
bud formation

1963 LAMPORT DTA. J. Biol. Acer pseudoplatanus:

Chern. 238:1438-1440 suspensions, hydroxylation
of proline in cell wall
glycoprotein

1965 LAMPORT DTA. Adv. Bot. Acer pseudoplatanus:

Res. 2:151-218 suspensions, review of
cell wall proteins with
emphasis on biochemistry
of hydroxyproline

1967 GIVAN CU & COLLIN HA. Acer pseudoplatanus:

J. Exp. Bot. 18: suspensions, changes in
321-331 respiration rate and
nitrogen content

1967 HOLLEMAN J. Proc. Natl. Acer pseudoplatanus:

Acad. Sci. 57:50-54 suspensions, incorporation
of proline into cell wall
glycoprotein

1967 LAMPORT DTA. Nature Acer pseudoplatanus:

(Lond.) 216:1322-1324 suspensions, nature of
hydroxyproline-rich
glycoprotein of cell
wall

1969 BROWN EG & SHORT KC. Acer pseudoplatanus:

Phytochemistry 8: nucleotide pattern in
1365-1372 suspension cultures

1969 LAMPORT DTA. Biochem- Acer pseudoplatanus:

istry 8:1155-1163 suspensions, nature of
cross-links between
hydroxyproline-rich
glycoprotein and poly-
saccharides in cell wall
 321

Year Author & Reference Species & Remarks

1969 SHORT KC, BROWN EG & Acer pseudoplatanus:

STREET HE. J. EXp. Bot. suspension, nucleic acid
20:579-590 metabolism

1970 DASHEK WV. Plant Acer pseudoplatanus:

Physiol. 46:831-838 suspensions, hydroxypro-
line rich glycoprotein in
cell walls

1970 DOREE M, LEGUAY J & Acer pseudoplatanus:

TERRINE C. C.R. Acad. suspensions, adenine
Sci. 270:2292-2295 metabolism

1970 LAMPORT DTA. Ann. Rev. Acer pseudoplatanus:

Plant Physiol. 21: suspensions, role of
235-270 proline, hydroxyproline
in cell wall

1970 SIMPKINS I, COLLIN HA Acer pseudoplatanus:

& STREET HE. Physiol. suspensions, growth
Plant. 23:385-396 response to nitrogen
sources

1970 SIMPKINS I & STREET HE. Acer pseudoplatanus:

J. Exp. Bot. 21:170-185 suspensions, effect of
kinetin on nitrogen
metabolism

1971 HEATH MF & NORTHCOTE DH. Acer pseudoplatanus:

Biochem. J. 125:953-961 callus and suspensions,
glycoprotein of cell
wall

1971 MATSUMOTO T et al. Populus (6 species):

Agric. Biol. Chern. 35: suspensions, nitrate
543-551 better nitrogen source
Chern. 35:543-551 than ammonium

1971 STUART R & STREET HE. Acer pseudoplatanus:

J. Exp. Bot. 22:96-106 suspensions composition
of conditioned media

1972 DOREE M, LEGUAY JJ & Acer pseudoplatanus:

TERRINE C. Physiol. suspensions, uptake of
Veg. 10: 115-131 adenine and leucine
related to CO 2 efflux

1972 NYGAARD P. Physiol. Acer pseudoplatanus:

Plant. 26:29-33 nucleotide pools
322

Year Author & Reference Species & Remarks

1973 COX BJ, TURNOCK G & Acer pseudoplatanus:

STREET HE. J. Exp. Bot. suspensions, uridine
24:159-174 metabolism

1973 DOREE M. Physiol. v~g. Acer pseudoplatanus:

11:267-290 suspensions, uptake of
adenine and substituted
adenine compounds

1973 DOREE M. Phytochemistry Acer pseudoplatanus:

12:2101-2108 suspensions, adenine
metabolism

1973 KING PJ, MANSFIELD KJ Acer pseudoplatanus:

& STREET HE. Can. J. suspensions, effect of
Bot. 51:1807-1823 nitrate on growth rate on
synchronous cultures in
chemostats

1973 MATSUMOTO T et al. Populus: suspensions,

Agric. Biol.-Chem. factors affecting antho-
37:561-567 cyanin production

1973 OZAWA T, HAGA K & Castanea: accumulation of

TAKINO Y. Nippon Nogei asparagine in galls
Kagaku Kaishi 47:627-
631

1973 RAYMOND P et al. Acer pseudoplatanus:

Biochem. Biophys. cAMP in suspension cells
Res. Commun. 53:1115

1973 YOUNG MJ. Expt. Bot. Acer pseudoplatanus:

24: 1172-1185 suspension, nitrogen
assimilation during
nitrogen limited growth

1974 BRIGHT SW & NORTH COTE Acer pseudoplatanus:

DH. J. Cell Sci. protoplast suspensions,
16:445-463 bromodeoxyuridine mutants

1974 KING PJ et al. Acer pseudoplatanus:

Planta 117:109-122 suspensions, thymidine
kinase and aspartate
transcarbamoylase activity
in synchronous cultures,
thymidine uptake

1974 STEWARD FC, ISRAEL HW Acer pseudoplatanus:

& SALPETER MM. J. Cell metabolism of proline by
Biol. 60: 695-791 cultured cells
 323

Year Author & Reference Species & Remarks

1975 HAHLBROCK K. Planta Acer pseudoplatanus:

124: 311-318 suspensions, nitrate up-
take, growth, and con-
ductivity changes in the
medium

1976 GATHERCOLE RW & Acer pseudoplatanus:

STREET HE. New Phytol. suspensions, isolation of
77:29-41 cells having high phenyl-
alanine-ammonia lyase
activity

1976 JESSUP W & FOWLER MW. Acer pseudoplatanus:

Planta 132:119-123 suspensions, use of
nitrate or glutamate as
sole medium nitrogen
sources, effect on growth
rate, cell number protein

1976 JESSUP W & FOWLER MW. Acer pseudoplatanus:

Planta 132:125-129 suspensions, effect of
nitrogen source on respir-
atory activity and carbo-
hydrate utilization

1976 KING PJ. J. Exp. Bot. Acer pseudoplatanus:

27:1053-1972 suspensions, chemostat
cultures with nitrate of
glucose as limiting nutri-
ent cell composition

1976 KING PJ. Plant Sci. Acer pseudoplatanus:

Lett. 6: 409-418 suspensions, urea as sole
nitrogen source

1976 WESTCOTT RJ & Acer pseudoplatanus:

HENSHAW GG. Planta suspensions, phenyla-
131:67-73 lanine-ammonia-lyase
activity of cells

1976 WILSON G. Ann. Bot. Acer pseudoplatanus:

(Lond.) 40:919-932 suspensions, chemostat
cultures with glucose or
nitrate as limiting
nutrient
324

Year Author & Reference Species & Remarks

1977 GONSALVES AN et al. Eucalyptus grandis:

In: Sharp, Larse~ callus, nitrogen sources
Paddock & Raghaven related to growth
(eds.) Plant cell and
tissue culture prin-
ciples and applications,
Ohio State Univ. Press,
Columbus

1977 JESSUP W & FOWLER MW. Acer pseudoplatanus:

Planta 137:71-76 suspensions, relationship
of nitrogen source to
carbohydrate metabolism,
nitrate or glutamate as
nitrogen sources

1977 KING PJ. J. EXp. Bot. Acer pseudoplatanus:

28:142-155 suspensions, effect of
nitrate on cell division
and protein synthesis in
chemostat cultures

1977 POPE DG. Plant Physiol. Acer pseudoplatanus:

59:894-900 suspensions, relationship
between hydroxyproline -
containing proteins of
cell wall and medium

1979 THOIRON B et al. Acer pseudoplatanus:

Physiol. Plan~ suspensions, uptake of
46:352-356 leucine, methionine and
adenine affected by gas
shock

1980 BARR J & NORDIN P. Acer pseudoplatanus:

Biochem. J. 192: biosynthesis of glyco-
569-577 proteins by membranes

1980 CURE WW & MOTT RL. Acer pseudoplatanus:

Plant Physiol. 65: suspensions, effect of
supplement 90 urea and nitrate on growth
rate

1981 EVERETT NP J. Exp. Acer pseudoplatanus:

Bot. 32:171-182 sensitivities of 2,4-D
independent cell cultures
to nitrogenous compounds
 325

11. CARBOHYDRATE UTILIZATION AND METABOLISM

TREVOR A. THORPE

1. INTRODUCTION
Although great progress is being made in the production of
photoautotrophic tissues in vitpo (e.g., see 10, 181), cultured
plant cells, tissues and organs, even those that turn green in
light, generally require a carbon source for successful in vitpo
culture (50, 51, 145). As a matter of fact a carbon/energy
source is considered one of the five classes of essential sub-
stances needed to bring about growth and organized development
in vitpo (36, 50).
The importance of a carbon/energy source in the medium has
been recognized for a long time. The pioneering studies with
excised root cultures in the 1930's (175) led to the examination
of the types of carbohydrates suitable for root cultures, their
mode of uptake, metabolism, etc. (145). The carbohydrate re-
quirements for callus culture were first studied experimentally
in the 1940's using carrot root callus (53, 54). These early
studies led to the conclusion that sucrose was the best carbon
source for in vitpo cultures, although a variety of other cou-
pounds could be utilized by plant tissues in culture.
The capacity to utilize various carbon sources for growth
and development in vitpo indicates that these (or their degrada-
tion products) must be taken up into the cells, converted to a
metabolizable form and integrated into cellular metabolism.
In the intact plant approximately 70% of the fixed C02 is util-
ized in cell wall biosynthesis. There is no evidence to suggest
that in culture most of the carbohydrate is not similarly in-
volved in cell wall biosynthesis. However, it will become
apparent that more information has been obtained on other as-
pects of metabolism using cell cultures. Although most of the
326

research carried out on carbohydrate utilization and metabolism

in vi t r o involve non-woody plants, it is believed that mechanisms
involved are the same for all higher plants. Thus this chapter
will contain much information that has been obtained from non-
woody tissue cultures.

2. NUTRITIONAL ASPECTS
Since sucrose is the sugar of transport in the intact plant
it is not surprising that this disaccharide and its constituent
hexoses, glucose and fructose generally best serve the carbon
nutritional requirements of cells in culture, including woody
tissues (6, 7). However a variety of other carbon sources have
supported some growth in culture (see 110, 145). The capacity
of tissues to utilize other carbohydrates varies with the species
from which the explant was selected (72). Furthermore, cultures
derived from different cultivars, e.g.,of Malus (22), different
organs of the same plant, e.g., root- vs. stem-derived callus of
sugar maple (113), and even single cell isolates from the sane
clone, e.g., of tobacco, have all been shown to differ greatly
in their response to a particular carbohydrate. Finally, if a
carbohydrate will support growth of a tissue in culture, it will
apparently also support its diff e rentiation (158, 170, 179).
Some selected examples of carbohydrate use, mainly in culture~

of woody species, are provided below. In addition to glucose

and fructose, other utilizable monosaccharides include galactose
(6, 61, 113), and mannose (6, 179). Disaccharides, including
maltose (101, 157, 179), cellobiose (113, 179), trehalose (113,
179), lactose (71, 115, 168, 185), and melibiose (61, 119) have
all supported growth in woody and non-woody plant species.
Raffinose, a trisaccharide, has been found to be suitable for
some species (61, 157, 179), as has the tetrasaccharide,
stachyose (61, 170). Polysaccharides, such as starch can also
serve as carbon sources (29, 119). Pentoses generally do not
support growth, although sugar cane and tomato can use ribose
(3, 119), and carrot xylose (57).
With respect to sugar alcohols, myo-inositol, although re-
quired in small amounts for successful culture of many plant
 327

species (e. g., 116, 178, 183) is unable to serve as the sole
carbon source. On the other hand, sorbitol supports excellent
growth of Malus tissue culture (23), as well as cultures of
several genera in the Roseae (25). However, it must be pointed
out that sorbitol is an important carbohydrate in the metabolism
of apple and related species in vivo. Mannitol supports callus
growth in Fraxinus (178). Glycerol can also be used by some
tissues (20, 56, 80, 136).
A specific morphogenetic role for carbohydrate has been
suggested by experiments carried out on the induction of vascular
elements in vitro. At a constant auxin concentration, low con -
centrations of sucrose (1.5-3.0%) favoured xylem formation in
lilac callus, while at higher concentrations (4.5-5.0%) ph lo em
was produced (174). Similar results were obtained with Phaseolus
callus (78). In liquid cultures of Parthenocissus callus the
number of xylary cells formed increased with sugar concentration
(up to 8%) (132). Sucrose also affected the quality of xylem
by altering the pattern of wall thickening (11). Low sucrose
concentrations (0.5%) induced elements with annular and scalari-
form thickening, whereas higher concentrations (1.5-3.5%) favour-
ed scalariform and reticulate elements. In Helianthus tuberosus,
sucrose and g lucos e were equa lly effective in differentiation
when used separately, but inhibitory when used together (114).
Althou gh it was once suggested that a -glucosyl disaccharides
possess special xylogenetic properties (78), it appears that
any carbohydrate which permits rapid cell pro liferation in a
particular tissue will effectively support xylogenesis (127).
From the above it is clear that tissues in culture can
utilize a variety of carbon sources for growth and organized
development . IIowever, rarely are any of these superior to
sucrose and its hexose constituents. For most cultures there-
fore sucrose at a level of 2-4% (w/v of medium) is the carbo-
hydrate of choice (50). It has been shown that the time at which
maximum yield is obtained coincides with the time at which carbo-
hydrate is exhausted from the medium (138, 167). However, all
the carbohydrate need not be added at the start of culture; if
some is added during log phase growth , it supports the same
328

additional growth as would have occurred, if the total carbo-

hydrate was added at the start of culture (167).

3. CARBOHYDRATE UPTAKE
In studying the uptake of exogenously supplied carbohydrates
a distinction should be made between those fueling primarily
exergonic pathways and those accumulated and utilized exclusively
for the endergonic synthesis of structural cell components (110).
Thus, most cells have been found to be freely perneable to glyco-
lytic intermediates, as well as sugars such as arabinose, xylose
and fucose, which do not promote growth but are components of the
cell wall. The major question which arises is whether the carbo-
hydrate uptake into cells is an active process, a passive dif-
fusional one or some combination.
Studies on sugar uptake into tobacco (122) and carrot callus
(125) and Acer c e ll suspensions (43) led to the idea that uptake
was passive. In contrast studies with sugar cane cells in sus-
pension indicated that the uptake of sugars was active because
it occurred against a steep concentration gradient, normal efflux
was not greater than 10 % of the influx rate, and uptake was in-
hibited by substances such as iodoacetate and dinitrophenol (106,
107). It was further concluded that specific transport sites
for glucose uptake were present on the cell membrane. Further
the almost instantaneous appea ranc e of sugar phosphates with
glucose uptak e suggested that the formation of phosphorylated
intermediates was an important part in the active uptake process,
which required a permease and a kinase in the plasmalemma (43).
Similarly, active uptake of maltose into variant strains of soy-
bean grown in cell suspension has been observed (101). The up-
take was inhibited by NaN 3 and dinitrophenol.
Whether uptake is passive or active, or a combination is
still not clear. However, it would appear that a distinction
has to be made between uptake into a tissue, i.e., into the
apoplastic portions (intercellular spaces, apparent free space,
etc.) and entry into cell. Improved techniques for the isolation
of membrane vesicles, vacuoles, etc., will allow this question
to be answered definitively. Indeed, some progress has been
 329

made through the use of protoplasts and isolated vacuoles. Use

of isolated vacuoles of red beet tissue (35) and pea mesophyll
cells (63), indicated clearly that the tonoplast was a seat of
the sugar transport system. Since the cytoplasmic component is
sDall relative to the vacuole, observed uptake of sugar into
isolated protoplasts (e.g.,62) could be accounted for mainly as
a result of active tonoplast uptake. However, detailed kinetic
studies, on initial uptake into pea mesophyll protoplasts have
suggested that passage through the plasmalemma was not due to
simple diffusion, but involved a specific transfer mechanism,
which was capable of discriminating between different sugar
isomers (64). Furthermore, based on transport inhibitor effects
in protoplasts kept in the light or dark, evidence was obtained
for the presence of at least two modes of energy-coupling for
sugar uptake into the plasmalemma, only one of which could be
accounted for by the proton-gradient model. Thus, based on the
above it appears that uptake of carbohydrates into cells is an
active, energy requiring process, while into the apoplast it is
passive.

4. CARBOHYDRATE METABOLISM
Studies on carbohydrate metabolism in vitro have been carried
out with callus cultures, cell suspensions and more recently with
continuous (chemostat) cultures.
In addition, to the metabolism of exogenous carbohydrates,
cultures grown in the light will contribute some photosynthate
to the endogenous carbon pool. For example, it was shown that
there was a greater increase in dry weight in a green strain of
rue over a colourless strain, as a consequence of higher absorp-
tion rates of sucrose from the medium and the refixation of res-
piratory C02 in the photoheterotrophic cells (139). Furthermore
dark or non-autotrophic C02 fixation occurred in culture (135,
154, 169). In spite of the presence of exogenous and endogenous
carbohydrates, it has been shown that CO 2 is essential for the
initiation of growth in cultured sycamore cells (52). The CO 2
requirement was not related to any effect on pH. The cells fixed
the C02 into organic and amino acids. This non-photosynthetic
330

C02 fixation was important in the synthe sis of Kr e bs cycle inter-

mediates. Even in photoautotrophically cultured cells 44% of the
total 14C0 2 fixation was in C4 compounds, particularly malate
(135) .
Carbohydrate metabolism has generally been studied in various
tissue culture systems by feeding the cultures with appropriate
radioactive sugar, followed by trapping of any labell ed C0 2
evolved, detection by various means of any labelled products
formed and by measuring the activity (or better, the capacity)
of various enzymes. In on e of the earliest studies using this
approach, it was shown in 1 954 that carrot callus readily inter-
converted glucose and fructose and synthesized sucrose when sup-
plied with either of the monosaccharid e s (55). Since then much
information has been obtained on carbohydrate metabolism in cul-
tured cells.
4.1. Sucrose degradation
Three major enzymes have been implicated in the metabolism
of sucrose in plant cells. These are invertase, sucrose phos-
phate synthetase, and sucrose synthetase. Plant invertases are
relatively complex, existing in the cell wall and in the cytoplasm
and having either acid or neutral to alkaline pH optima (43, 110)
It has been clearly shown that acid invertase is present in the
cell wall of carrot in vitro (165), since 60% of the total inver -
tase activity could be released from cultur e d cells by incubating
them in c e ll wall solubilizing enzymes. In addition, synthesis
and release of the enzyme into the medium occurred from the
protoplasts after cell wall removal.
In normal cell and callus cultures invertase is released
into the medium, initiating extracellular hydrolysis of sucros e
(142, 159). The rol e of cell wall invertase is not clear. With
the exception of sugar cane (110) and Japanese morning glory
callus (73), most tissue s examined could tak e up sucrose without
prior hydrolysis. Although a positive correlation has b ee n found
between cell wall invertase activity and the growth rate of Acer
cells (31), no such correlation was found with most other tissues
including tobacco (159), carrot (37), Convolvulus (95) or Ephedra ,
Cupressus funebris and Libocedrus decurrens (142). Use of
 331

asymetrically labelled 14C-sucrose has clearly demonstrated that

uptake of sucrose in carrot callus occurred without prior hydrol-
ysis (124). A more recent study confirmed the lack of correla-
tion between growth rate and acid invertase activity in carrot,
but suggested that the level of acid invertase controlled the
endogenous levels of sugars which, by interaction with phyto-
hormones, affected both morphological and biochemical differen-
tiation (126). Thus the extracellular hydrolysis of sucrose is
not required for its uptake in most systems.
~he intracellular breakdown of sucrose occurs via soluble
invertase and sucrose synthetase (43, 110). In Acer (31) the
soluble invertase activity paralleled growth rate, while in
tobacco (159) and Japanese morning glory (74) sucros e synthetase
seemed more important. In the last species the change in activity
for sucrose synthetase was greater than that for sucrose phosphate
synthetase. This latter enzyme has not been extensively examined
in cultured cells.

4.2. Metabolism of other carbon sources

It is generally accepted that carbon sources, other than
glucose and fructose, must be converted into either of these
hexoses for integration into metabolism. The utilization of
pentoses such as ribose is not clear. Presumably they enter
directly into metabolism on being phosphorylated. However, in
very few cases, have the necessary biochemical studies been
carried out with cell cultures to demonstrate that the presuned
route of metabolism does indeed take place in culture.
The utilization of exogenous starch has been shown to involve
extracellular hydrolysis by amylases, which were secreted into
the medium; e.g., in J u nipe r u s (28), sugar maple (112) and
Cu pre ssus (143). Both a - and S-amylases were released by sugar
cane, and their contents in the medium were greatest in the
presence of starch (104). Similarly the utilization of the tri-
saccharide raffinose and the disaccharide maltose have been shown
to be dependent on the secretion into the medium of the appro-
priate hydrolytic enzymes (110, 181) or their presence in the
cell wall e.g., a -glucosidase (94). It should be pointed out
332

that invertase can degrade raffinose. In sugar cane cells,

a-galactosidase was also important in raffinose utilization.
However, the level of free galactose remained small (108), sug-
gesting its rapid mobilization (see below). a-Galactosidase was
also detected in cucumber hypocotyl explants and callus cultured
on the galactose-containing sugars, stachyose, raffinose,
melibiose (61). Only sucrose, fructose and glucose accumulated
in the cells, but the free-space sugars varied; therefore, it
was suggested that the a-galactosidase was located in the cell
wall.
Growth of tissues on lactose has been linked with the sub-
strate induction of B-galactosidase (71, 115, 168). The enhanced
activity, e.g., 5-fold increase in cotton, did not persist in the
absence of the sugar (115). The increase in enzyme levels was
inhibited by actinomycin C 1 and cycloheximide in Nemesia and
Petunia (71), indicating the requirement for both RNA and protein
synthesis. Since in most tissues galactose is toxic if it ac-
cumulates, it is to be presumed that the galactose released in
the cells is rapidly metabolized.
In addition to a - and B-galactosidases and B-glucosidase,
B-xylosidase and a -mannosidase have been detected in isolated
cell walls of Convolvulus (128). This indicates carbohydrate
containing appropriately linked xylose and mannose can be cleaved
extracellularly.
The utilization of galactose by sugar cane cells was apparent-
ly dependent on a 35% increase in galactose-kinase activity and
a 10-fold increase of UDP-galactose-4-epimerase activity (108).
This enhanced enzyme activity allowed for the ultimate production
of glucose-l-P for entry into intermediary metabolism. The
apparent key to galactose utilization was the enhanced level of
the epimerase, which prevented the accumulation of UDP-galactose
in the cells.
The utilization of sorbitol and other cyclitols involve their
conversion to the corresponding hexoses. In the case of sorbitol,
sorbitol dehydrogenase, which reversibly converts sorbitol to
fructose, has been detected and characterized in apple callus
tissue (117). The presence of other polyol dehydrogenases has
 333

not been reported in cultured tissues.

4.3. Hexose mobilization and metabolism

The hexoses most commonly metabolized are glucose and
fructose, and to a large extent initial mobilization of different
carbon souces will lead to formation of these hexoses directly
or derivatives thereof such as phosphates or nucleotides.
The normal entry of glucose into oxidative metabolism is via
glucose-6-P, a reaction catalyzed by hexokinase. The entry of
fructose is not as clear, although the formation of fructose-6-P
via hexokinase catalysis is probably the route used. Hexokinase
therefore regulates the flow of carbohydrate into both glycolysis
and the pentose phosphate pathway. The relative contribution of
these two pathways to carbohydrate oxidation has b een measured by
the C 6 /C l ratio; the further the deviation from unity, the greater
being the contribution of the pentose phosphate pathway to the
oxidative process. In terms of energy production, the oxidation
of pyruvate, arising from glycolysis, is carried out in the
mitochondria through the tricarboxylic acid cycle which is coupled
to oxidative phosphorylation in the process of respiration. The
interme diates of the TCA cycle are also important in providing
carbon skeletons for other aspects of metabolism. Resulting
from the presence of sucrose synthetase in cells, sucrose is
broken down to UDP-glucose and fructose. The production of this
sugar nucleotide is important in starch as well as cell wall
biogenesis. The functioning of these various pathways in cul-
tured tissues has been examined and will be discussed in relation
to growth and organized development in vitro .
4.3.1. Cell cycle studies. Using populations of synchron-
ously dividing cells of Acer pseudoplatan us (89) and Vinca rosea
(96), it has been found that there were peaks of respiratory
activity at the S and late G2 phases. The activities per cell
of two key enzymes of glycolysis, phospho-fructokinase and
pyruvate kinase and enzymes of the TCA cycle, malate dehydrogenase
(96) and succinic dehydrogenase (89) followed a similar pattern.
Adenylate energy charge (a relative measure of the ATP to ADP and
AMP levels - a useful measure of metabolic activity) decreased
334

from the M to the GI phase, reached a minimum at GI/S and then

increased gradually (96). The activities of key enzymes of the
pentose phosphate pathway, glucose-6-P dehydrogenase and
6-phosphogluconate dehydrogenase, decreased during the M phase
to a low level in the GI phase but increased in S phase and re-
mained at a high level during the G2 phase in Vi n c a cells (96) .
A similar pattern of activity of glucose-6-P dehydrogenase was
found in Ace r cells (89). The C6/CI ratio had a peak at the end
of the M phase and then decreased during GI' and showed low values
during Sand G2 phases (96).
The peaks in respiration at the GI/S to Sand G2 phases in-
dicated that energy was required for the RNA and/or protein
synthesis which occurred during those periods. The activity of
the pentose phosphate pathway was high during the Sand G2 phases
and therefore indicated a requirement for a supply of intermed-
iates and reducing power during these phases.
4.3.2. Growth studies. Cell growth depends on carbohydrate
utilization for two primary functions, viz: formation of major
cell components and as an energy source. The balance between
utilization for these two purposes varies with the stage of
development (110). Growth studies have been carried out using
callus, batch suspension and continuous (chemostat) cultures.
In dark-grown tobacco callus, the endogenous content of
reducing sugars initially increased but by day 9 in culture it
had decreased to less than 50% of the day 3 maximum (160). Growth
rate varied in the light and the dark in the presence or absence
of gibberellic acid (GA3) and in the presence of various carbon
sources (157). However these differences in growth rate and
final weight after 5 weeks in culture could not be correlated
with differences in the rate of glucose oxidation as judged by
the specific activities of enzymes of the glycolytic and pentose
phosphate pathways, although there was a light-dependent increase
in the activities of glucose-6-P dehydrogenase and 6-phosphoglu-
conate dehydrogenase beyond day 14 in culture.
Neither light- or dark-grown tobacco callus accumulated
starch in the presence or absence of GA3 (161). However there
was a higher rate of starch turnover in light-grown tissues, as
 335

judged by the specific activities of the synthetic and degradative

enzymes of starch metabolism. GA 3-treated tissues contained a
lower level of starch and this decrease could be correlated with
the specific activity of phosphorylase. It would appear, there-
fore, that since the differences in growth rate were not a con-
sequence of carbohydrate oxidation or starch metabolism, they
might be related to the fraction of glucose available for cell
wall biosynthesis. Certainly the cell wall composition of
tobacco callus varied with the growth rate as influenced by
cytokinin level in the medium (68).
Sugar maple callus stored starch which was rapidly hydrolyzed
in response to temperature (111). A decrease of 40% in the
starch content occurred when the tissue was exposed to 4 C for
48 h. The decrease was less at higher temperatures, but no
decrease in starch was observed at 3 C or at 27 C. Furthermore
a change of 1 C between these extremes, markedly affected the
starch-sugar balance. The most abundant sugars present in the
tissue were glucose, fructose and sucrose. Thus the callus
tissue responded in culture like the intact plant.
In cell suspensions respiratory measurements follow more or
less the same pattern (110). 0 2 consumption increased two fold
or more until the 4th to 6th day in culture, this was followed
by a period of reduced oxygen demand over the subsequent 8-14
days. Measurement of the activities of the glycolytic and pen-
tose phosphate pathways and the C6/Cl ratio in Ace r cells indi-
cated that both pathways were enhanced during the early stages
in culture, but following the initiation of cell division, car-
bohydrate oxidation was predominantly via the glycolytic pathway
(41). In agreement with the above, isolated mitochondria from
Ace r had the highest cyanide-sensitive respiration during the
active cell division phase (177). It has also been shown that
the pentose phosphate pathway made a greater contribution to
glucose catabolism during the initial stages in culture of Vi n ca
cells than did glycolysis (82). This initial relative enhance-
ment of the pentose phosphate pathway could be interpreted as
indicating a higher requirement for NADPH and pentoses for
reductive biosynthesis and nucleic acid metabolism. Indeed,
336

enhancement of pathways involved in pyrimidine nucleotide bio-

synthesis occurred during the cell division phase in Vinca (82).
In confirmation of the above, major increases in the level of
NADPH, ATP and energy charge have been observed during the lag
phase of culture in Ace r cells (137). Thus it appears that cells
and tissues with high growth rates generally have a high activity
of both the pentose phosphate pathway and glycolysis, whereas
cells with a low growth rate oxidize carbohydrate predominantly
via glycolysis.
The nitrogen source has also been shown to influence the
activities of these pathways in Ace r cells (79). Cells grown on
N03 as opposed to glutamate grew faster and had higher levels
of pentose phosphate and glycolytic pathway activity. The major
difference was in the activity of the pentose phosphate path~my.

This finding indicates the probable demand for reducing power for
N03 reduction.
In a study on the effect of oxygen supply on the growth of
tobacco cells in suspension under aerobic-dark batch culture
conditions it was found that a very low value for the volumetric
02-transfer coefficient was sufficient to produce 14.9 g bio~ass

(dry weight) from 35 g sucrose (85). In a further study under

02-limited conditions, true growth yields of 107 g cell dry
weight/mol glucose and 61 g cell dry weight/mol 0 2 were obtained
(84). Furthermore it was calculated that the carbon-substrate
consumed by the cells was metabolized mainly in biosynthetic
processes, without the excretion of extracellular products. Such
studies are important in maximizing yield per unit cost particu-
larly when cell cultures are being used on a large scale, e.g.,
as in chemostat conditions, commercially.
Continuous chemostat cultures, which allow for the maintenance
of steady states of cellular activity, have been used to study
the regulation of carbon flux in Ac e r cells (42, 43). Further-
more, this approach has been used to study carbon metabolism
during the transition from one steady state to another. A com-
parison of carbohydrate oxidation under three specific growth
rates (cell doubling times) indicates that at low and medium
growth rates the pentose phosphate pathway enzymes had low
 337

activities relative to equivalent glycolytic enzymes (43, 90).

However at high growth rates this situation was reversed. Fur-
thermore, there were increases in the level of 0 2 uptake and
also a net increase in the total carbon flux. How is this change
in metabolism brought about during the transition period? Based
on the analyses of the activities of the carbohydrate oxidation
pathways, measurement of key metabolites, sucrose utilization
and oxygen uptake, it appears that the key feature of the
behaviour of the cells was the pronounced oscillations in all
parameters measured except sucrose utilization (42-45). There
was an initial small decrease in the rate of sucrose utilization,
but there was a dramatic change in the pattern of utilization,
in that the % involved in terminal oxidation decreased from 60%
to 35% before rising again to about 62% towards the end of the
transition period. The rate of 02 uptake increased as the level
of hexose consumed through terminal oxidation rose. Changes in
enzyme activity took longer to occur than the changes outlined
above. Pentose phosphate pathway enzyme activities increased
earlier than the glycolytic enzymes, apparently in response to
the need for reducing equivalents and pentoses. As the growth
rate approached the new steady state there was a significant
dampening of the oscillations. Similar type oscillations have
been observed in a stepdown situation (reduction of input N03 in
N03-starved cells) and in a transition from N03 to glucose
limitation (88). The significance of these oscillations in
relation to regulation of metabolism remains to be determined.
4.3.3. Organized development. Carbohydrate metabolism in
relation to two areas of organized development, viz: embryogenesis
and organogenesis, will be examined.
Although the role of carbohydrate in vascular tissue formation
has been known for a long time (see Section 2), no studies have
apparently been carried out on carbohydrate oxidation during xylem
or phloem formation in vitro. However, the recent establishment
of a single cell system from isolated mesophyll cells of Zinnia
el egans, which differentiates directly into tracheary elements
in large numbers without intervening cell division (47, 48), will
permit the carrying out of such biochemical studies in relation
338

to the energy and other requirements for xylogenesis.

Studies on carbohydrate metabolism during somatic embryo-
genesis suffer the same fate as most studies carried out on this
process. The reason is that the normal approach used is to trans-
fer embryogenic clusters from an auxin-containing medium (in
which the embryogenic cells are induced) to an auxin-free medium,
in which embryo development occurs. Thus it is not clear if the
metabolic changes observed are directly associated with embryo-
genesis or simply result from the absence of (a release from)
auxin (154). Nevertheless it has been shown that embryogenic
carrot cells are rich in starch (146), which disappeared from
these cells during embryo formation. Embryogenic cells were also
rich in mitochondria.
Higher mitotic activity, a lower starch content and intensi-
fied respiration was observed in carrot cells in auxin-free
medium (33). This increased cell division has been confirmed
(173). Dissolved 0 2 concentration influences embryo development
in carrot (86). Below a critical level embryogenesis occurred,
but above that level rhizogenesis was favoured. The lowered 0 2
concentration led to an increase in cyanide-sensitivity and
higher cellular levels of ATP. The level of ATP in the cells
could also be raised by adding exogenous adenosine at higher 0 2
concentrations. Both treatments increased somatic embryo develop-
ment.
Enzymatic and other biochemical studies on carbohydrate
metabolism during somatic embryogenesis have apparently not been
carried out. Part of the reason is no doubt due to the lack of
a suitable experiment system. However recent methods for obtain-
ing embryogenic material at different morphological stages (172)
and the induction of partial synchrony in the early stages of
embryo development (46) should permit such studies to be under-
taken in the near future.
Changes in the starch content of organ-forming tissues have
been observed in several plant species (13, 26, 103, 164). In
shoot-forming tobacco callus the peak of starch accumulation
occurred just prior to the formation of meristemoids (158,163).
The accumulation of starch occurred in the presence of several
 339

different carbon sources in light or dark-grown cultures (158).

Ultrastructural studies carried out with Datu r a innoxia showed
that starch accumulation, in the form of polysaccharidic granules,
began on the first day in culture (14). The inhibitory effect of
GA 3 on organogenesis in tobacco, rice and tomato leaf discs has
been linked with a reduction in the accumulation of starch in the
early stages of organ initiation (26, 103, 164). However the
reversion by GA3 of primordia already formed to callus could not
be accounted for by its effects on starch metabolism (162).
Examination of the activities of enzymes involved in starch
metabolism in shoot-forming tobacco callus revealed that the
accumulation of starch resulted mainly from increased synthesis
throughout the culture period. The reduction in starch during
meristemoid and shoot primordium formation involved enhanced
rates of degradation (160, 161). Compared to proliferating
callus tissue, higher levels of soluble and insoluble starch
synthetase and Q-enzyme were observed throughout the culture
period. In contrast, activities of R enzyme and a -amylase were
higher only in the later part of the culture period, whereas the
activity of phosphorylase fluctuated. There were no differences
in the activities of UDP-glucose and ADP-glucose pyrophosphoryl-
ases between shoot-forming and callus-proliferating tissues (123).
This indicates that the regulation of starch accumulation was
not at the level of sugar nucleotide synthesis. A continuous
supply of free sugars from the medium was required for shoot
formation (151), and while there was no difference in the uptake
of 1 4 C-sucrose into shoot-forming and non-shoot-forming tobacco
callus, there was a steady and linear incorporation of 1 4C into
starch in the former tissue (15).
The rate of respiration was found to be higher in shoot-
forming tobacco callus during shoot initiation and in the shoot-
forming lower half of the callus than in non-organ-forming upper
part of the callus (134, 158). Similarly, higher rates of res-
piration have been found in cultured cotyledon explants of
radiata pine, which are undergoing shoot initiation (12). In-
creased activities of both glycolysis and the pentose phosphate
pathways were observed in shoot-forming tobacco callus, compared
340

to proliferating callus (156). Furthermore higher levels of

activity were found in the lower (shoot-forming) half of the
tobacco callus. During meristemoid formation the shoot-forming
tissue metabolized 14C-glucose at a higher rate (1.7x) than non-
organ-forming tissue, but based on C 6 /Cl ratio both pathways of
carbohydrate oxidation were equally enhanced. These findings
indicated a higher energy requirement as well as greater needs
for NADPH and pentoses for biosynthesis for shoot initiation
than for callus growth.
Studies on non-photosynthetic C02 fixation showed that the
activities of enzymes involved in malate metabolism, viz: phos-
phoenol pyruvate carboxylase, malic dehydrogenase, glutamic-
oxalacetic transaminase and malic enzyme, were generally higher
in shoot-forming tobacco callus compared to proliferating callus
(129). While there was a continuous accumulation of malate in
growing callus (also found in suspension-grown Acer cells, 169)
shoot-forming tissue showed a dramatic increase early in culture,
followed by a rapid decline during organ initiation. It there-
fore appears that the primary role of malate metabolism in shoot-
forming tissue was the production of NADPH via NADP-linked malate
enzyme. Measurement of NADPII and NADP+ pools showed that there
was a faster decline and more complete utilization of NADPH and
a greater build up of NADP+ levels in shoot-forming than non-
shoot-forming tissue (17). These findings confirm the apparent
need for reducing power for the organogenetic process.
Total adenosine phosphates in the shoot-forming tobacco
tissue increased during the early culture period (1.6 X at day 6)
and declined thereafter (17). However the levels in shoot-
forming tissue were always higher than in callus-proliferating
tissue. NAD+ levels were higher in shoot-forming tissue, but
the level of NADH was low in both shoot-forming and callus-forming
tissue. The energy charge of the shoot-forming tissue declined
the most, and reached its lowest value during meristemoid for-
mation. Isolated mitochondria showed a trend towards higher
and more efficient respiration, as well as lower levels of
cyanide-insensitive respiration (19). These findings support
the idea that organ initiation is a high energy requiring process.
 341

These types of studies on carbohydrate metabolism have been

carried out on only one organ-forming callus system, i.e., tobacco.
Nevertheless, the findings may well be general, in view of the
requirements for energy and reducing power for many aspects of
metabolism.

4.4. Cell wall biogenesis

As indicated earlier a major portion of fixed carbon is used
for the synthesis of the cell wall in higher plants. Cell and
protoplast cultures have proven useful tools for the study of
cell wall biosynthesis. To attempt to review all of the infor-
mation available on this topic is beyond the scope of this
chapter. Nevertheless, because of the importance of this topic
for forestry research, some aspects of cell wall studies carried
out in vit r o will be briefly discussed. These include some
aspects of both primary and secondary cell wall formation and
cell wall polysaccharide turnover.
4.4.1. Primary cell walls. The primary cell wall of plants
consists of microfibrils of cellulose embedded in a complex
matrix composed of polysaccharide, prot e in and glycoprotein (38).
Traditionally the polysaccharides of this matrix have b e en
separated and classified on the basis of their solubilities.
Thus pectic substances are soluble in water, in chelating agents
or in dilute acid. The hemicellulosic components require alkali
for solubilization. The cellulose microfibrils are insoluble in
these solvents. The primary cell wall can be considered as a
network consisting of discrete polysaccharides and protein linked
together by covalent and noncovalent bonds (99). It has become
clear, particularly in the last decade, that the cell wall is not
an inert passive structure, but a dynamic entity in which some
turnover is occurring and which contains functional enzymes.
These are involved not only in cell wall metabolism, but playa
vital role in the functioning of the rest of the cell.
Many of the present ideas on the organization of the polymers
in the primary cell wall have resulted from studies using suspen-
sion-grown cells of Acer (2). The most important advantages of
the suspension cultured cells for these studies are that firstly,
342

they can be grown as a relatively homogeneous tissue possessing

primary and no secondary walls; and secondly, the cells secrete
into the culture medium polysaccharides that are similar in
composition to the noncellulosic polysaccharides of the cell wall.
Notwithstanding the advances being made in the understanding of
many aspects of the primary cell wall, our knowledge is still
primitive with many unanswered questions (99). Furthermore
there is no clear consensus on the actual organization of the
primary cell wall, as the different analytical methods used give
different interpretations.
The composition of the primary cell wall of suspension-
cultured sycamore cells is as follows: rhamnose (3.1 a ), fucose
(1.3), arabinose (21.0), xylose (7.6), mannose (0.3), galactose
(12.8), non-cellulosic glucose (3.7), cellulosic glucose (23),
galacturonic acid (13.4), total protein (10), hydroxyproline (2),
(2). These components are linked together in polymers as follows:
arabinogalactan (20%), cellulose (23%), protein (10%), rhamno-
galacturonan (16%), tetraarabinosides (9%) and xyloglucan (21%).
Together these give a classical composition of pectin - 34%,
hemicellulose - 38% and cellulose - 20%; values which are typical
for primary cell walls. For details on structure and composition
of the primary cell wall, reference can be made to the following
reviews (2, 5).
The synthesis of carbohydrate polymers in cell walls involves
the transfer of sugars from their sugar nucleotide derivatives
to the growing polysaccharide chains. Starting with glucose the
various sugar nucleotides can be synthesized (39, 83). These
include ADP-, GDP- and UDP-glucose; UDP-galactose, -glucuronic
acid, ~rabinose, etc. Some sugar nucleotides also arise from
myo-inositol (83, 102).
The pectic substances consist of a complex mixture of acidic
and neutral polysaccharides (38). The acidic portion of these
pectins consists of a 1-4 linked polygalacturonans, which con-
tain various sugars. The carboxyl groups of the galacturonic
acid residues are methylated to varying degrees. The neutral

aweight % composition
 343

fractions are ~ade of polysaccharides containing apiose, arabin-

ose, rhamnose, xylose, etc. (30). Studies on the biosynthesis
of pectic substances have shown that UDP-galacturonic acid is
the most active glycosyl donor to the a 1-4 polygalacturonic
acid backbone of the acidic pectins, on which methylation takes
place (38). Pulse chase experiments have shown that the for~ation

of the neutral components and their incorporation into pectins is

complex and varies with the stage of growth and differentiation
(30, 144).
Hemicelluloses are mainly composed of D-glucose, D-galactose,
D-mannose, D-xylose and L-arabinose joined together in different
combinations and in various glycosidic linkages (38). They also
contain glucuronic and other uronic acids. The major hemicellulo-
sic components of cell walls of monocots and dicots are the
arabinoxylans and xyloglucans respectively. The xyloglucans,
B 1-4 glucan with a 1-6 xylose side chains, have been postulated
to cross link with the cellulose fibrils and other non-cellulosic
polysaccharides to stabilize the cell wall (2).
Cellulose, B 1-4 glucan, is a polymer which make s up fro~ 1/3
to 1/2 of the dry weight of terrestrial plants. In green plants
it now appears that glucose is transferred from UDP-glucose (and
also from GDP-glucose) to form a lipid-pyrophosphate-glucose and
then probably a lipid-pyrophosphate-cellobiose compound (27). The
cellobiose residue is subs e quently split off to be attached to a
water-soluble transient intermediate polymer of glucose or of a
glucose derivative. These oligosaccharides are apparently linked
to protein (75), and then go on to form cellulose microfibrils.
Initial polymer formation in cellulose biosynthesis is assoc-
iated with the membranes of the Golgi bodies, but cellulose
microfibril formation occurs on the plasmalemma (32), where the
nascent polymer is transferred outside by the process of exo-
cytosis. The glucosyltransferases involved in pectic and heni-
cellulosic polysaccharide synthesis are also associated with the
membranes of the Golgi complex (121).
Glycoproteins ar e also a constitue nt part of primary cell
walls. Some of these are enzymes e.g., peroxidase, and some
others have surface-binding capacity, i.e., are lectins (38).
344

A common feature of these glycoproteins is the presence of

N-acetylglucosamine. Sugars such as galactose, arabinose and
mannose are commonly involved in the linkage. These cell wall
proteins tend to be rich in hydroxyproline. Furthermore, the
Golgi apparatus and dictyosomes are involved in the biosynthesis
and secretion of these glycoproteins.
Protoplasts have proven useful in the study of cell wall
biogenesis (40, 49). Cell walls generally appeared within 24 h
in culture and were initially cellulosic of short polymer length.
Cellulose with normal polymer length was formed later. During
the early stages of cell wall formation the matrix polysaccharides
were lost to the medium. Glycoprotein formation also took place
at that time. These studies were found to support the idea that
cell wall formation always preceded cell division in cultured
protoplasts.
The addition of various cell wall constituents and precursors
such as arabinose, xylose, galactose, mannose and glucuronate
to the medium of sucrose-grown tobacco cellsrlid not stimulate
growth of the cultures (70), indicating that none of these were
rate limiting for cell wall biosynthesis. However myo-inositol
caused a slight stimulation. In carrot cell suspensions the
synthesis of myo-inositol from glucose took place in culture
(171). Furthermore, label from myo-inositol was also found in
arabinose, xylose and galacturonic acid residues isolated from
the cell wall polysaccharides.
4.4.2. Cell wall turnover. The composition of the primary
cell wall changes with culture condition and medium and also
with age of the culture. Thus, for example, the induction of
friability in compact tobacco callus by low cytokinin content in
medium in dark-grown cultures is accompanied by changes in the
cell wall composition (68). Friable callus had a greater extensin
content and a higher arabinose/xylose ratio in the hemicellulose
fraction~ Suspension cultures of Vinca rosea show 3 distinct
growth phases: a cell division phase, a cell expansion phase and
a stationary phase (147). During these phases there were de-
creases in polyuronide and non-cellulosic glucan contents and
increases in xyloglucan and a -cellulose contents. Stationary
 345

phase cells of Ac e r had a higher hydroxyproline content than

actively dividing cells (133). These findings indicate a
strengthening of the cell wall with age in culture.
Different cell wall components undergo different rates of
turnover in culture. Thus a comparison of cell wall and extra-
cellular polysaccharides in the medium of suspension-grown cells
of Vinca r o s ea revealed that the rate of synthesis of pectic
substances was higher during the cell division than cell expansion
phases of cell growth, as would be expected (148). There was
little turnover of the pectic polysaccharides or of the cellulosic
fraction, but an active hemicellulosic turnover during these
phases. The major hemicellulosic turnover was found to be in the
arabinose and galactose units and not in the xylose or glucose
units (149). This suggested that the main turnover was in the
3-6 linked arabinogalactans, and not in the xyloglucans. This
was confirmed by pulse chase experiments (150), which showed
that the polymers composed of arabinose and galactose were most
actively synthesized, deposited in the cell wall, rapidly degraded
and secreted into the medium. Since hydroxyproline-rich protein
has been found linked to arabinogalactan in the cytoplasm (76,
130), it was suggested that the sugar polymer acted as a carrier
for extensin into the cell wall, where hydrolysis would lead to
the deposition of the extensin in the cell wall and the release
of the arabinogalactan into the medium. Presumably in the intact
plant the hemicellulosic fraction would be retained within the
cell wall.
The highest activities of cellulase and polygalacturonase
in Rosa gla uc a suspension cultures were observed at the end of
the exponential growth phase of the cells (81). This indicates
that hydrolysis of the pectic and cellulose fractions in the cell
wall plays a role in cell maturation.
4.4.3. Secondary cell walls. The cytodifferentiation of
cells leading in particular to the formation of secondary cell
walls is an important feature of all vascular plants. In woody
taxa the accumulation of xylem is a consequence of this process
in which lignin along with cellulose become the dominant com-
ponents of the cell wall. Lignin contributes about 20-30% of dry
346

weight of the woody sterns of trees, making it the second most

abundant natural product after cellulose (60).
Lignin is a high molecular weight compound, formed by the
polymerization of substituted cinnamyl alcohols; the phenyl-
propanoid units being p -coumaryl, coniferyl and sinapyl alcohols.
Conifer lignins have been more intensively investigated than any
others (60). Spruce lignin is composed of the above alcohols
in a ratio of ca. 14:80:6. This ratio 'is cornmon to softwood
lignins, which are, therefore, mainly composed of guaiacyl
residues. In contrast dicot lignins are copolymers of both
guaiacyl and syringyl units in approximately equal amounts.
The lignin macromolecule is not simply deposited within the
polysaccharide matrix of the cell walls, but it is bound to car-
bohydrates via covalent linkages (98). One important polysac-
charide-lignin linkage is the carbohydrate p-hydroxybenzyl
ethers. Ester bonds arising from the peroxidase catalysis of
carbohydrates and ferulic acids or its condensation product also
occur (176).
The aromatic amino acid, phenylalanine, is generally recog-
nized as the main precursor for phenylpropanoid biosynthesis.
This amino acid arises from carbohydrates via the shikimate
pathway (see Section 4.5.). The sequence of reactions involved
in the production of the cinnamyl alcohols is outlined in Fig. 1.
All of the enzymes involved have been isolated from lignifying
tissues of gymnosperms and angiosperms, and they have pronounced
specificities towards phenylpropanoid substrates (60).
The final step in lignin biosynthesis is the polymerization
of p-coumaryl, coniferyl and sinapyl alcohols. This reaction
sequence is initiated by the oxidation of the phenolic hydroxyl
group of these monomers, yielding mesomeric phenoxy radicals.
Coupling of these radicals leads to the formation of dilignols
which on reoxidation and repeated radical coupling lead to the
formation of oligomeric intermediates and finally combine to form
the lignin macromolecule (60). The cell wall peroxidase/H 2 0 2
system plays an important role in these reactions in lignifying
tissue (69).
In vitro culture s have proven useful in elucidating some of
 347

HO - o C H 2 -CH(NH 2 )
Tyrosine
- COOH o CH 2 -CH(NH 2 )
Phenylalanine
- COOH

~----- H0-oCH'CH-COOH OCH'CH-COOH

p- Coumoric acid Cinnomic acid

-0-
CH 3 0

H 0 - o - CH' CH - COOH -- H0-o-CH.CH-COOH -- HO CH CH - COOH

HO Caffeic acid CH 3 0 Ferulic acid CH 30 Sinapic acid

I I I
I I I

+ t t
L - -~ P - Coumaryl alcohol Coniferyl alcohol Sinapyl alcohol
I I :
I I I

:L ________________ ~'
I
LIGNIN 4-------------------
~
: I

FIGURE 1. General flow chart showing conversion of aromatic

amino acids to cinnamyl alcohols, the building blocks of lignin.

the steps outlined above. Thus work with parsley cell suspension
cultures have shown that the coordinated activation of the enzymes
involved in phenylpropanoid metabolism is preceded by RNA and
protein synthesis (67).
Lignin synthesis in tobacco cell cultures has been studied in
cell aggregates (97). Larger aggregates produced more tracheary
elements and had higher activities of shikimate dehydrogenase,
cinnamic acid 4-hydroxylase, caffeic acid-O-methyltransferase, and
5-hydroxyferulic acid-O-methyltransferase. The peak of activity
occurred just prior to tracheid formation. Similar changes have
been observed during xylogenesis in bean callus (65).
The activity of the pentose phosphate pathway was elevated
during lignification in Jerusalem artichoke and Coleus blumei
tissues relative to glycolysis (131). The enhanced activity of
the pentose phosphate pathway indicated that it was probably
providing the reducing power needed for lignification, in addition
to erythrose-4-P (see Section 4.5.). The development of an
active lignifying system in Zinnia elegans (47, 48) will allow,
348

as indicated earlier (section 4.3.3.), the study of both carbo-

hydrate and phenylpropanoid metabolism during secondary cell wall
development.

4.5. Carbon skeleton utilization

During oxidation of carbohydrate via glycolysis or the pen-
tose phosphate pathway, followed by further oxidation of pyruvate
in the TCA cycle, carbon can be lost from these cycles for other
metabolic purposes. As indicated earlier one function of the
pentose phosphate pathway is the production of pentoses for
nucleic acid metabolism. In addition, erythrose-4-P, along with
phosphoenol pyruvate from glycolysis are the precursor carbo-
hydrates for the shikimate acid pathway. This pathway leads to
the production of the aromatic amino acids, phenylalanine,
tyrosine and tryptophan. Similarly the carbon skeletons of the
other common amino acids are derived from only a few metabolic
intermediates, mainly from the TCA cycle. Fatty acids arise
also from carbohydrate via pyruvate in the form of acetyl-CoA.
Thus all major aspects of metabolism dip into the carbohydrate
pool for their precursors. In the intact plant, carbon fixation
(both photosynthetic and non-photosynthetic), the pentose phos-
phate pathway, glycolysis and the TCA cycle all contribute to
this pool. Under most cell culture conditions, carbohydrate
(usually sucrose) in the medium and non-photosynthetic CO 2
fixation provide the carbon needed.
Nonautotrophically fixed C0 2 (from 1 4C-bicarbonate) was
shown to be incorporated into lipid, protein, amino acids and
organic acids in Paul's scarlet rose after 90 mins incubation
(118). Approximately 20% of the C02 taken up was stored in the
above compounds, the rest being presumably released. In log
phase cells a higher proportion of the 1 4C entered protein,
whereas in stationary phase cells malate accumulated. Malate
accumulation in stationary phase Ac e r cells has also been re-
ported (169). The amount of carbon drained from the TCA cycle
for protein synthesis varied with age of the cell culture of
Paul's scarlet rose (77). During the period of most rapid
protein synthesis 1/6 as much carbon was drained as was released
 349

as C02. During stationary phase cells only one-thirtieth was

drained.
Shoot-forming tobacco callus has been shown to have a higher
level of shikimate pathway activity than non-shoot-forming
tissue (9). Two to four fold increase in activity of 3-deoxy-D-
arabino-heptulosonate-7-phosphate synthetase (the coupling
enzyme), shikimate kinase, chorismate mutase and anthranilate
synthetase were found. Presumably therefore higher rates of
tryptophan, phenylalanine and tyrosine production were occurring
during organized development. The significance of the activation
of the pathway remains to be determined, but the increased
phenylalanine production could be important for lignification
of the vascular tissue in the newly-formed shoots (see section
4.4.3.) .
The contribution of carbohydrate metabolism to other aspects
of metabolism in cultured cells remains a relatively unexplored
area of research.

5. OSMOTIC ROLE
The major component of a tissue and cell culture medium is
usually the carbohydrate. For most species sucrose best serves
this purpose (see section 2.). As indicated earlier (section
4.1.) sucrose can be taken up without hydrolysis or some
hydrolysis may occur extracellularly and the products taken into
the cell. In any case the intracellularly hydrolyzed sucrose
increases the osmotic potential in the cell, unless the products
of hydrolysis are rapidly removed by metabolic utilization.
Furthermore, the optimum level of sucrose (or other carbon source)
in the medium, is in excess of the requirements of the tissue
for growth, and relatively large quantities of sucrose and
reducing sugars accumulate eventually (151, 159).
Osmotic potential is regulated in plant cells by both in-
organic ions and organic molecules, including organic acids,
sugars and sugar alcohols (184). It has been suggested that
because the cytoplasmic inorganic ion concentration remains
fairly constant, osmotic adaptation of the cytoplasm is mainly
achieved by the accumulation of non-toxic organic molecules (180).
350

Knowledge of turgor-triggered processes is very limited (184).

However, there is experimental evidence to support the idea of
a direct control of membrane transport and the electrical prop-
erties of the cell membrane by turgor pressure. There is also
evidence that turgor-controlled processes play an important role
in the regulation of cell expansion (24). According to this view
auxin gives the cells sufficient wall-loosening capacity to permi
cell enlargement, which will only occur if the necessary driving
force, turgor pressure, is available. Therefore the ability of
cells to generate osmoticum, and thereby obtain sufficient water,
will determine the rate of cell enlargement.
Osmotic effects of the medium on growth and differentiation
are rarely considered, even though the average tissue culture
medium, e.g., Murashige-Skoog salts with 3% sucrose has a water
potential of ca. -5 bars. Indeed the effect of an increase in
medium water potential from near zero (1/2 strength White's
mineral salts) to -1 bars significantly reduced the rate of
growth of wound callus from cut ends of Fraxinu s excelsio r (34).
At near zero water potential the callus was characterized by
very active surface growth and little internal differentiation.
At potentials greater than -1 bar, the callus had suberized
surfaces and contained lignified xylem and sclereids. While at
near zero water potential callus appeared after 2-3 days, it did
not appear until after 6-9 days in medium of -6 to -10 bars.
Subcultured callus and inde ed most explants require a medium
in which solutes (minerals, reduced N, carbohydrate, etc.) must
be present for successful growth and differentiation. Thus,
under in vitr o culture conditions, the cells are exposed to wat e r
potentials similar to those of normal field-grown plants, which
may experience a range in soil water potential from zero, after
irrigation, to -15 bars at drought. The effects of mild and
more severe stress on tissues in culture have been examined.
Stress is usually imposed by the addition of non-electrolytes
such as carbohydrate, sugar alcohols or polyethylene glycol
(carbowax). Very mild stress (addition of PEG 1000 at 1 % w/v,
which increased water potential of the medium 0.5 bars) caused
an increase in tobacco callus osmotic potential, r e duced growth,
 351

as well as decr e ased sucrose, fructose and glucose contents of

the tissue. It had no effect on the water content of the callus,
but increased its dry matter (92). More pronounced stress fur-
ther increased the above parameters, and in addition reduced
the water content of the tissue. Reduction of the relative
humidity of the air above the cultures from 90 to 32% increased
the osmotic potential and the dry weight, as a % of the fresh
weight, and sugar content of the tissue (93). In soybean, in-
creased medium water potential led to a decr e ase in cell size,
the cells becoming spherical, and an increase in callus produc-
tion (fresh and dry weight) (87). Small increases in osmotic
stress (-2 bars) decreased growth of sugar cane cells in culture
(109). The greater medium water potential also caused increased
respiration, a high turnover of amino acids, a lower reducing
sugar and elevated sucrose content, and reduced invertase
activity. Furthermore, the effects were greater in cells not
preconditioned to the greater medium wat e r potential.
High sucrose levels have been us e d succ e ssfully for Ln vitro
embryogenesis (4), as well as the culture of zygotic embryos
(120). In the latter cultures the younger the embryo that was
excised the higher the concentration of sugar that was required
(182). In cultured barley embryos interactions between hormone
levels and osmotic potential have been observed (58). Growth
regulators which favored the formation of callus led to the
accumulation of starch and a decrease in cellular osmolarity.
These effects were not observed if shoot development occurred.
High levels of sucrose in the medium (ca 8% w/v) were also
found to be optimum for rooting of Pinus lambertiana embryo cut-
tings in vitro (59) and sugar cane cells (105). In P. l am be r-
tiana, osmotic substitutes, applied alone or in combination with
sucrose, could not duplicate its effect on root reg e neration.
These findings are in keeping with the well known horticultural
practice of selecting cuttings rich in stored starch and free
sugars for rooting (e.g., see 66). In the latter cases it is the
presumed requirement of carbohydrate for energy which allows
root primordium formation to occur. However part of the carbo-
hydrate could be acting colligatively.
352

The optimum concentration of sucrose in the medium is 3%

(w/v) for growth and shoot formation in dark-grown tobacco cal-
lus (16). A reduced number of shoots were obtained when lower
or higher levels of sucrose were incorporated in the medium.
aowever cultures grown on 2% sucrose (or above) gave the same
number of shoots, if supplemented with mannitol to give the same
medium water potential as 3% sucrose. Below 2% sucrose, addition
of mannitol, although giving the same medium water potential as
3% sucrose, could not substitute for sucrose; neither could
mannitol at any level serve as energy source for growth.
Increased levels of bacto-Agar in the medium, while lowering the
medium water potential, did not promote shoot formation or re-
place carbohydrate (and mannitol) for that process. This indi-
cated that part of the carbohydrate in the medium (1/3) was
acting in an osmoregulatory role and that the osmoticum must
enter the tissue. Other studies with different genetic lines of
light-grown tobacco callus cultures showed that the optimum
sugar content in the medium differed for growth, greening and
shoot formation (8). Improvement in number of shoots and their
morphology was associated with a reduction in the sucrose content
of the medium, whose water potential was maintained at control
levels by the addition of mannitol. Further studies (18) showed
that shoot-forming tobacco callus, grown in a medium with the
same water potential as non-shoot-forming tissue, maintained
greater (i.e., more negative) water and osmotic potentials, as
well as greater (more positive) pressure potentials than non-
shoot-forming tissue. These differences were observed by day 2
in culture, which is prior to any visible histological changes
ln the tissue, and were maximum at day 6, which is at the time
of the first visible changes leading to organized development.
These differences were maintained throughout the culture period.
They did not result from differences in the uptake of sucrose
from the medium. In addition, fresh weight/dry weight ratios
and water content of the tissues were of the same order. Similar
differences were also found in tissues grown under low or high
sucrose levels, although they formed fewer shoots. Mannitol
substituted for carbohydrate gave the same tissue potentials and
 353

maintained the differences observed between shoot-forming and

non-shoot-forming tissue. It was suggested that the greater
water, osmotic and pressure potentials were maintained by (a) the
accumulation of malate early in culture, (b) the accumulation of
free sugars from the medium throughout the culture period, and
(c) the degradation products of starch at the time of meristemoid
formation and the succeeding organogenetic phases (see section
4.3.3.) .
In the formation of adventitious shoots in vitro in explants
or callus, it is at times necessary to transfer the buds which
are induced to another medium for the development of shoots (e.g.,
see 152, 153, and chapter 4 in this volume). This is particu-
larly true in conifer tissue culture, where the second medium is
often devoid of growth regulators. However in some cases a
reduction of sucrose, e.g., in radiata pine, from 3% to 2% (1)
and alfalfa, from 3% to 1% (139a) was necessary. It is probable
that the inhibition of shoot development in the presence of high
levels of carbohydrate lS partly osmotic. Similarly, in rooting
of adventitious shoots in conifers, reduction of the mineral
content of the medium by half is common (e.g., see 152, and
chapter 4 in this volume), and in some cases the level of sucrose
for optimum rooting is low, e.g., 0.5% in Douglas fir (21). Here
again osmotic effects could be involved.
The use of carbohydrate in osmotic studies suffers from the
drawback that it is readily metabolizable. Other osmotic agents
such as the polyethylene glycols are generally considered to be
non-metabolizable and not to penetrate into the tissue, although
they may enter under certain conditions (100). For some tissues
PEG is toxic but this can often be eliminated by removal of metal
ions from the PEG through dialysis or passage through ion exchange
columns (141). For some cultures even those treatments are not
enough to avoid toxic effects. Inhibition of shoot formation in
tobacco callus was observed in the presence of low concentrations
of PEG which barely reduced the medium water potentials (16).
Furthermore, at higher concentrations (e. g., 1% w/v PEG 200) the
agar containing medium did not solidify. Comparative studies
with PEG and mannitol in sugar cane cells indicated that the PEG
354

effect was more pronounced at the same medium water potential

(109), indicating possibly a slight toxic effect.
Mannitol does not support callus growth for most plant
species (see section 2, and 72, 145). However, it is thought
to enter the tissue (91) but not to be actively metabolized,
except in Fraxinus and some other members of Oleaceae (165).
Certainly the capacity of Fra x inus pennsylvanica callus to grow
on mannitol as the sole carbon source (178) would indicate that
it must enter the cells. Thus it appeared that if mannitol is
used instead of sucrose to fulfill the osmotic requirement for
shoot formation in tobacco, it acted as an intracellular osmotic
a gent (16). An as yet incomplete examination of the uptake of
14 C-mannitol into suspension cultures of carrot and tobacco cells
and cultured excised cotyledons of radiata pine has indicated
that carrot and tobacco c e lls absorbe d less than 5 % and radiata
pine about 10 % of the mannitol available over a 14 h period
(Thomp son, Douglas and Thorpe, unpublished). However, in tobacco
about 50 % of the mannitol which was taken up was metabolized;
ca. 4 % was r e leased as CO 2 , ca. 30 % was converted to an as yet
unidentifi e d low mol e cular we ight sugar or other neutral compound
and a furth e r ca. 15 % was converted into basic, acidic and in-
soluble compounds. Similar findings have been obtained with
carrot and radiata pine. In the latter speci e s some adaptation
to mannitol apparently takes place, as an e nhanced rat e of man-
nitol degradation occurred in tissue cultur e d with mannitol
prior to exposure to the labe lled sugar alcohol. Thus the
osmotic eff e ct of mannitol apparently takes place from outside
th e cell, i. e ., from mannitol in the cell wall, intercellular
spaces, etc., i. e ., in the apoplast. In any cas e , much of the
sucros e plac e d in the me dium also remains outsid e of the cells.
Wh e th e r or not the osmotic agent is outside the plasmal e mma or
insid e the tonoplast, its osmotic e ffects on organe lles in the
cytoplasm is likely to be the same . The efficiency of mannitol
as an osmotic age nt thus apparently is a consequ e nce of its low
uptak e into cells.
The significance of the osmotic adjustments for growth and
organized deve lopment r e mains to be det e rmined. However it is
 355

reasonable to assume that biophysical events playas important

a role as biochemical ones. The biophysical events may involve
changes in membrane properties and the biochemical events pre-
sumably include a shift in metabolism (155). One possible
consequence of the increased osmotic potential of shoot-forming
tissue is the enhancement of the activity of the mitochondria
for energy production (Brown and Thorpe, unpublished). Isolated
mitochondria from tobacco were found to b e more efficient in ATP
production when assayed at the greater osmotic potentials found
in shoot-forming tissue than at those found in the non-shoot-
forming callus.

6. CONCLUDING THOUGHTS
In this chapter I have discussed various aspects of carbo-
hydrate utilization and metabolism relative to in vitro cultures.
Studies on the nutritional capacity of different carbon sourc es
can better be carried out with cell, tissu e and organ cultures,
than with intact plants since most cultures are nutritionally
heterotrophic. These studies tend to indicate that plant cells
in culture are very plastic and possess the capacity to utilize
a variety of carbon sources, in addition to fixing C02 non-
photosynthetically. Use of protoplasts and cell suspension
cultures have gone a long way towards resolving the problem of
active versus passive uptake of carbohydrates. While uptake into
the tissue is indeed passive, it now appears fairly certain that
uptake into the cell, i.e., across the plasmalemma is an active
process, which is relatively unspecific.
The relatively recent indication of an osmotic component to
shoot initiation should stimulate conc e rted efforts to evaluate
the role of other physical phenomena in differentiation. Work
done in the 1960's (e.g., see 140) has certainly shown a con-
tributory role for pressure in organized development. In
addition, the recent acceptance of the membrane as a focus for
the regulation of a large segment of a cell's b e haviour, lends
importance to studies in this area.
As an advance on the use of tissue slices, cell and tissue
cultures have proven extremely useful tools in the study of
356

metabolism. Investigations on carbohydrate metabolism have

indicated quite convincingly that in vitro cultures utilize both
primary pathways of carbohydrate oxidation to satisfy their
requirements for energy, reducing power and to procure carbon
skeletons for other aspects of metabolism. Use of cell cultures
and protoplasts are allowing for a better understanding of the
composition and biosynthesis of the primary and secondary cell
wall, including the regulation of phenylpropanoid metabolism.
While more information is presently available with respect to
growth, new data are emerging on aspects of organized development
in vitro. Based to a large extent on the findings obtained with
Ac e r cells, it is apparent that the utilization and metabolism
of carbohydrate is similar in woody and non-woody plants. Thus
the findings reported here for non-woody plants are likely to be
obtainable also with woody plants. In conifers, the involvement
of starch metabolism in organized development is likely to be
less than in angiosperms, due to the fact that lipid (rather
than starch) is the predominant reserve polymeric substance.
Nevertheless other aspects of carbohydrate metabolism are likely
to be same.
Various aspects of carbohydrate metabolism need to be
examined further and some of these have been indicated in the
text. In particular, studies on xylogenesis, organogenesis and
embryogenesis should be undertaken with woody plant material.
Unfortunately to date success in regenerating plants via embryo-
genesis in woody species is very limited. Thus the bulk of the
investigations on plant regeneration will be based on primordium
formation and subsequent organogenesis in explants. No good
xylogenic system exists with woody material at present, and this
could hinder some of the biochemical studies needed to be
carried out on secondary wall formation. The same is true, to
a lesser extent however, with respect to primordium formation
in woody plants in general and conifers in particular. The
recent development of a conifer shoot-forming system, using
cotyledon explants of radiata pine (1) promises to be a very
useful tool for examining many physiological and biochemical
aspects of organogenesis, including studies on carbohydrate
 357

metabolism, due to the large number of shoot primordia formed.

Recent reviews on fundamental aspects of organogenesis and
embryogenesis (153-155) indicate clearly that slow progress is
being made in understanding the regulation of differentiation.
Nevertheless based on the rate of progress in the practical
exploitation of in vi t ro culture in the latter half of the past
decade, one can be reasonably optimistic that more researchers
will begin to examine the basic aspects of growth and organized
development using tissue culture systems, including woody species.

Acknowledgements. The author acknowledges with gratitude the

contribution of colleagues, associates and students to the
personal research reported here; as well as the discussions
held with Michael R. Thompson and David W.M. Leung on some of
the papers cited in this article. I also wish to thank Laura
Bentley for her valuable assistance in assembling and organizing
the references and proof-reading the article, and Erin Smith
for skillfully typing the manuscript. Financial support
for research from the Natural Sciences and Engineering Research
Council of Canada (formerly the National Research Council) and
the University of Calgary is also gratefully acknowledged.

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180. WYN JONES RG, R STOREY, RA LEIGH, N AHMAD, A POLLARD 1977
A hypothesis on cytoplasmic osmor eg ulation. In E Marre,
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368

185. ZRYD JP 1978 Induction of glycosidase activity in

sycamore cells. Abstracts IAPTC Congress. Calgary, p 174
 369

12. THE USE OF IN VITRO TECHNIQUES FOR GENETIC MODIFICATION OF FOREST TREES

E. G. KIRBY

1. INTRODUCTION
Encouraging reports of the development of techniques for in vi t r o mutant
selection (56) and for fusion, culture and regeneration of somatic hybrid
plants from protoplasts of herbaceous species (79) suggest applications of
new techniques in genetic improvement programs for forest trees. In
addition, through a more thorough understanding of gene expression in
higher organisms (9), genetic engineering of plants, including forest
species, is likely to become a reality in the near future (47). Previous
reviews have addressed the topic of somatic cell genetic manipulation of
plants (26, 28, 56, 72, 78, 79, 83). The present paper will discuss the
use of in vitro systems for genetic modification of forest trees. It must
be noted at the outset that recent contributions are few in number. Con-
s iderable additional effort must be expended before use of such techniques
becomes an integral part of tree breeding programs.

2. IN VITRO SELECTION
Alterations in established culture procedures for selection of specific
variants in a population of microorganisms is a standard procedure (14).
Considerable interest has centered on the application of similar procedures
to plant cells. The ideal s ystem for such application consists of tech-
niques enabling indefinite culture of friable callus, complete regeneration
of whol e plants from cell suspensions or callus cultures resulting from
s uspensions , production of haploid cell lines from pollen culture or other
t echnique and stab i liz ation of the chromosome karyotype (72). This review
will discuss significant progress which has been made with regard to sev-
eral of these prerequisites of a model system for forest species, although
considerable work remains for full rea lization of in vitro procedures.
370

2.1. Natural variation

In order to select for a specific variant a significant amount of
genetic variability is required within the cell population to which the
selection is applied. A simple direct selection procedure may involve
exposure of a population of cells to a mutagenic or cytotoxic substance
followed by isolation of cell lines that are resistant to a specific
treatment, such as low temperature tolerant lines, drug resistant lines,
or lines with the ability to utilize unusual amino acids or carbohydrates
(56). All selections either in vivo or in vitro focus on phenotypic
characters. Both epigenetic and direct genetic factors determine pheno-
type and it is essential when isolating variant lines to determine if the
source of variation is indeed genetic or epigenetic in nature. Many
examples of epigenetic variation in plant cell cultures are found in the
literature, the most thoroughly investigated being cytokinin and auxin
habituation (7, 61, 62). Drug resistance, for example cycloheximide, has
also recently been shown to be epigenetic (35, 57). On the other hand,
a number of traits which have been selected in vitro have been sexually
transmitted, demonstrating the genetic nature of selected phenotypic
variation. Such studies, as reviewed by Maliga (56), indicate that use
of in vitro selection procedures followed by regeneration of plants from
variant cell lines can offer a powerful tool to tree breeders.
A surprising degree of phenotypic and genetic variation has been
reported in protoplasts isolated from assumed clonal leaf material of
potato (81). Shepard suggested the use of clonal cell lines derived from
single protoplasts (protoclones) as a source of genetic variation of
practical significance, as in the case of pathogen resistance in potato
(81). In tree species there is some indication of naturally-occurring
phenotypic variation expressed in vitro. Huhtinen's group (43, 44) was
able to produce plantlets from cambial callus of an early-flowering geno-
type of birch (Betula pendula). Normally, plants of this genotype flower
in the first year of growth and produce only flowering shoots at the time
of flowering. A number of plantlets regenerated from cambial callus pro-
duced vegetative shoots simultaneously with flowering shoots, signifying
a phenotype not expressed in the explant material. Further studies are
needed to determine the nature of this phenotypic change. It appears
that natural variation may be readily expressed by tree species in vitro.
It remains a challenge for geneticists working with such species to devise
 371

adequate selection procedures.

2.2. Induction of variation

The frequency of genetic variation in populations of plant cells grow-
ing in vitro may be increased by treatment with mutagenic substances or
exposure to radiation, procedures which alter the primary base sequences of
DNA. The efficiency of various mutagenic treatments in increasing genetic
variation in cultured plant cells has been reviewed (56). Substances in-
cluding ethylmethane sulfonate, N-methyl-N-nitrosoguanidine, e thylenimine,
N-ethyl-N-nitrosourea as well as treatments with ultraviolet and X-irradia-
tion have been used to increase genetic variation in protoplasts, cell
suspensions and callus cultures of a number of species. The use of mutagen
treatments generally increases the rate of spontaneous mutation by two to
one hundred fold (15, 16).

2.3. Selection techniques

Briefly defined, three techniques are utilized for identifying the
appearance of variant phenotypes in cultures of plant cells. Direct
seZection, likely the most frequently employed, involves isolation of cell
lines that have the ability to grow under specific conditions. Most
variant clones isolated to date are resistant to drugs or amino acid analogs
and several procedures for direct isolation and characterization have been
proposed (15, 16, 23, 56, 57, 58, 91, 91). As an alternative to direct
selection, indirect seZection is utilized when the desired phenotype does
not permit continued growth on the se lective medium. Indirect selection
in vitro is not to be confused with indirect selection used in breeding
programs, where genetic improvement of a specific trait is accomplished by
selection based on a genetically correlated trait. An excellent example of
indirect selection in vitro is illustrated by the selection of nitrate
reductase deficient lines of tobacco by exposing cell populations to chlor-
ate, a nitrate analog (65). In cells producing nitrate reductase, chlorate
is reduced to chlorite, which is toxic. Cells producing low levels of
nitrate reductase, or none at all, are not killed and are, therefore, in-
directly selected. Another indirect selection technique, l arge ly adopted
from mammalian tissue culture, involves the use of bromodeoxyuridine (BUdR)
(12) and flurodeoxyuridine (FUdR) (3).
It is not always necessary to develop an elaborate procedure for
372

selection of variant clones. This is especially true after treatment with

mutagens, where a small fraction of a wild type population survives the
treatment. Survivor clones are simply screened for appropriate phenotypes.
Such procedures have proved adequate in the isolation of auxotrophic mutants
in plants (8, 74, 78).

2.4. Plant regeneration

A prerequisite for utilization of any variant cell line produced in
culture for plant improvement is a reproducible procedure for regenerating
whole plants . If cell suspensions are utilized for selections, a plating
technique permitting callus formation from single variant cells, i.e., a
cloning procedure, must be developed. The most widely used procedure, the
Bergmann technique (6) involves suspending cells in a warm agar medium.
Plates are then poured and after the medium has solidified, individual
proliferating colonies are easily isolated. Various modifications of this
procedure have been described. Weber and Lark (90) have developed an
efficient plating procedure for isolating mutant lines of soybean and Datupa
using a l ayer of feeder cells to stimulate growth. For plating both proto-
plasts and cell suspensions of Douglas-fir a system involving the use of a
non -woven polyester fabric support has been developed (50, 51). This allows
for gas exchange and rapid diffusion of inhibitory substances, both of which
may limit growth of protoplasts of cells of conifer species when plated on
agar media. Figure 1 shows the proliferation response of a protoplast-
derived cell culture of Douglas-fir when plated on a fabric support . The
non-woven nature of the fabric is clearly visible.
The ability to regenerate economically important variant lines from cell
and callus cultures is crucial to their application in breeding programs.
Embryogenetic as well as organogenetic pathways for plantlet regeneration
in vitpo have been addressed in this volume for both gymnosperm (Chapter 4)
and angiosperm (Chapters 5, 6 and 7) tree species. With continuing intensive
research focusing on the fundamental challenge of achieving high rates of
embryogenesis and organogenesis in cell cultures of important tree species,
we can expect in vitpo selection to become feasible and practical. It should
be noted, however, that mutagenic treatments have many effects on plant cells,
one of which may be a loss in the potential for organized growth (22).
 373

FIGURE 1. Cell culture derived from protoplasts of Douglas-fir plated on

non-woven polyester fabric support.

2.5. Applications
There are many applications for variant cell lines in tree breeding
programs, including establishment of resistance clones, such as those
resistant to low temperatures, fungal pathogens including blister rust,
Dutch elm disease, fusiform rust and Melampsora rust (94) and also those
resistant to various insect pathogens. To date, however, little headway has
been made in this potentially significant area. In terms of screening
genotypes for rapid growth, several laboratories have found a good correlation
between in vitro and in vivo performance. Examples include correlations
between field performance and callus growth in aspen callus cultures (59) and
frequency of adventitious bud formation in cotyledon cultures of conifers
(1, 64). Tissue cultures may be utilized for screening a variety of tree
species for effects of environmental stresses including low temperatures in
spruce (87) and susceptibility to fungal pathogens, such as Dutch elm disease
in ulmus (86). Advantages of in vitro genotype testing include the short
time required to obtain results; tests may be able to be performed on
seedling-derived cultures; in vitro screening tests generally require only
minimal space; tests can be performed at any time of year. In our laboratory
investigations are currently in progress determining the usefulness of
several biochemical markers of cell cultures as a means of early genotype
374

assessment.
A few words of caution, however, are needed. Under most circumstances
conditions bn vitro do not simulate conditions under which plant cells
normally grow bn vivo . The mere act of placing plant mat eria l in culture
exposes cells to continuous selection pressure resulting in an increased
population of cells that are adapted to specific culture conditions (72).
In addition, differences in performance in vitro vs. in vivo may not be a
consequence of genetic differences, but rather a result of one or several
epigenetic factors controlling phenotype, as discussed earlier.

3. SOMATIC HYBRIDIZATION
Breeding programs for trees and crops are often faced with the major
problems of interspecific and intraspecific incompatibility which prevent
genetic recombination between unlike plants. Consequently, the first report
of the use of cell wall hydrolyzing enzymes for the isolation of large
numbers of naked protoplast s of higher plants (18) coupled with availability
of a procedure enabling high frequency of protoplast fusion (48) and culti-
vation procedures (11) have sparked considerable interest in production of
somatic hybrid plants. Several recent reviews (11, 48, 89) have focused on
principles and applications of protoplast techniques to crop improvement.
The discussion here centers on protoplast research with regard to potential
applications in tree breeding.

3.1. Protoplast techniques

Protoplasts of conifer forest species were first isolated in 1974 by
Winton's group from callus cultures of Douglas-fir (93), although the first
successful culture of conifer protoplasts was not reported until several
years later (21, 51). From thes e reports, sev eral factors appear essential
for the isolation of large numbers of protoplasts from conifer tissues.
The physiolog ica l condition of the starting material from which protoplasts
are prep ared appears critical. Douglas-fir cotyledons, after culture on an
agar medium containing cytokinin and auxin yielded large numbers of proto-
plasts (Fig. 2) when compared with numbers of protoplasts isolated from
cotyledons cultured on media without growth regulator s or with lower levels
of auxin (50).
Protoplasts are generally prepared from suitable plant material by
treatment with a mixture of cellulase and pectinase enzymes suspended in a
 375

FIGURE 2. Protoplasts freshly isolated from cotyledons of Douglas-fir.

plasmolyzing solution to ensure protoplast integrity. Several enzymatic

treatments have proven successful for isolation of conifer protoplasts.
David and David determined that integrity of mononuclear protoplasts isolated
from cotyledons of maritime pine (Pinus pinaster) could be maintained by a
long-term incubation of cotyledon material in a low concentration, (0.16% w/v)
of a mixed enzyme solution (21). We have found that rapidly dividing proto-
plasts can be prepared from pre-cultured cotyledons of Douglas-fir by short-
term treatments (two hours) using a more concentrated enzyme solution
(2.5% w/v) (51). In addition, to minimize osmotic stress to physiologically
fragile conifer protoplasts, especially during short term incubations at
high enzyme concentrations, it may be necessary to carefully adjust the
level of the osmotic stabilizing agent, sorbitol or mannitol.
Duhoux (25) has observed that aseptically isolated pollen grains of
Arizona cypress (Cupressus arizonica) will lose the exine layer upon hydra-
tion. The cellulosic intine of such pollen grains is then readily digested
with cellulase resulting in production of haploid protoplasts. Attempts to
culture such pollen protoplasts have been unsuccessful. Such techniques,
however, may be instrumental in development of haploid material for genetic
studies.
Limited reports are available concerning successful culture of proto-
plasts of conifer species (21, 50, 51). Review of these reports reveals
several factors that affect production of dividing cells from conifer proto-
plasts. First is the nature of material from which protoplasts are prepared.
To date there are no reports of successful culture of protoplasts isolated
from conifer suspension or callus cultures. In fact, successful culture
376

appears closely tied to the young, seedling origin of cotyledonary material.

In addition, work on both maritime pine and Douglas-fir (21, 51) has shown
that a pre-culture or pre-treatment of cotyledonary tissues with growth
regulating substances prior to protoplast isolation is required in order to
regenerate mitotically active cells.
Media for successful culture of conifer protoplasts (21, 51) contain
simple basal components plus mannitol or sorbitol as osmotic stablilizers,
sucrose, auxin and cytokinin and organic additives. Initially a complex
medium modeled after that of Kao and Michayluk (49) containing yeast extract,
serine, glycine, nicotinic acid, pyridoxin and biotin, was developed for
cotyledon protoplasts of Douglas-fir (50, 51) (Figs. 3A and B). This
medium has since been greatly simplified and callus cultures can be obtained
from Douglas-fir protoplasts by culture in basal medium containing sucrose,
growth regulators, sorbitol and high levels of glutamine. The glutamine
requirement for cell division has triggered a series of further studies in
our laboratory on regulation of nitrogen assimilation enzyme levels in vit~o.

fIGURE 3. a) Initial stages of cell proliferation in protoplast cultures

isolated from Douglas-fir cotyledons. b) Later stages of proliferation
showing many dividing cells. Protop1asts were cultured on a modified Kao
and Michayluk medium (50).

Another factor which has aided our ability to regenerate dividing cells
from conifer protoplasts is use of a non-woven polyester fabric support, as
described earlier. fabric supports and similar such procedures may aid in
gas exchange and diffusion of inhibitory substances from actively growing
cells.
 377

Among angiosperm forest species protoplast techniques offer promise.

Redenbaugh et al. (75) have isolated protoplasts from pollen mother cells,
tetrads and microspores of American elm (Ulmus americana) in attempts to
use protoplast fusion products to overcome sexual incompatibility. To date,
however, plantlets have not been produced from protoplasts of either gymno-
sperm or angiosperm forest species. The potential for tree protoplasts as
agents for somatic hybridization is striking. Because of incompatibility
problems, extremely long life cycles and short term viability of pollen of
some forest species, somatic hybridization techniques may offer hope for
tree breeders.
Many questions, however, must be answered with regard to somatic
hybridization by anyone attempting to utilize the procedures developed
recently. Is a selection system necessary whereby hybrid plants are pheno-
typically recognized? At present, the vast majority of reports of selection
systems developed for somatic hybrid plants are based on previous knowledge
of sexual hybrids. As techniques become available for production of plant-
lets from callus of tree species and somatic hybridization becomes an
applicable technique, new systems for precise selection of hybrid cells or
plantlets will be required. This alone is one of the important challenges
facing applied developmental geneticists.
Additional applications of protoplast research should be mentioned,
including utilization of protoplasts for the production of hybrid cytoplasm
and for introduction of foreign organelles and subcellular particles (11).
Exciting applications are apparent. Such procedures circumvent problems
of mitotic synchrony of fusion partners in heterokaryocytes and potential
nuclear incompatibility. Cytoplasmic characters may be utilized which have
demonstrated value in plant improvement. Proquction of cytoplasmic fusion
products ("cybrids") has been demonstrated in the tobacco system (32, 33,
96). Work from Power's laboratory (46) has demonstrated the usefulness of
cytoplasmic hybrids produced via inter-specific protoplast fusion in the
transfer of male sterile cytoplasm in the genus Petunia. Incorporation of
such procedures into breeding programs is likely to follow.
Considerable interest has been generated regarding the use of fusion
procedures for transfer of subcellular particles. Fusion can be induced
between freshly isolated protoplasts and isolated organelles or microorganisms.
The majority of studies in this area report genetic complementation by up-
take of isolated chloroplasts (27). A number of fundamental studies have
378

been performed (13, 63, 82). Considerable work rema in s and some doubt has
been expressed as to whether isolated chloroplasts, when incorporated into
foreign cytoplasm, are capable of surviving and expressing the chloroplast
genome (27).
One aspect of protoplast work that may have application to t r ee species
is the use of in vitro systems for the e stablishment of nitrogen f ixation
(19 , 29). Progre ss ha s been made with r e gard to the establishment of the
symbiotic association in vi t ro (4, 5, 10,42). Giles and Whitehead (30, 31)
report successful transfer of both cells and nitrogen fixin g ability of
Azotobacter vinelandii to protoplasts of the fungu s Rhizopogon . Thi s
specific fungus forms mycorrhiz a l associations with the roots of radiata
pine (Pinus r adi ata). When the nitrogen-fixing variants of Rhizopogon wer e
associated with roots of nitrogen starved seedlings of radiata pine, an in-
crease in growth and nitrogen levels in seedling material was observed.
Such studies demonstrate potential usefulness of functional symbiotic gene
transfer.

3.2. Graft hybridization

Interest has been generated concerning us e of graft hybr i dization, as
an alternative to protopl a st procedures, for the production of somatic
hybrids (69, 70, 71). Theoretically, during the process of preparation of
stock and scion partners in a graft, cell walls of cells at the cut surfaces
are mechanically removed. The juxtapositioning of the two partners results
in cell fusion. If such fusion takes place under conditions that favor
callus and adventitious bud formation, expression of hybrid charact ers may
be observed. Treatment of cut surface s with cellulases and the use of
polyethylene glycol to enhance fusion frequency may aid in hybrid production.
Thorough systematic studies of such procedure s, however, are needed and the
hybrid nature of resulting variants must be demonstrated.

4. GENETIC TRANSFORMATION
4.1. Principles
Isolation, culture and plant regeneration from protoplasts have enabled
the combination of genes from diverse sources. On the other hand, genetic
transformation involves uptake of selected DNA molecules by competent cells,
integration of the DNA into the genome of those cells and ultima te expres-
sion of integrated foreign genetic information (84) . Studies of genetic
 379

transformation in higher plants must be carefully scrutinized. It is not

the aim of this review to evaluate the many attempts at genetic transforma-
tion, but rather to briefly discuss techniques which have been described
and to point out their potential use in tissue culture of forest tree
species.

4.2. Procedures
4.2.1. DNA uptake. Since the original demonstration, almost thirty
years ago, that bacteria could be genetically transformed by exogenously
supplied DNA (2), considerable work in plant systems has focused on simple
uptake studies. Some controversy has developed in terms of the results
that have been obtained. Work from Ledoux's laboratory (54, 55), claiming
correction of a thiamine-less mutant in Arabidopsis as a result of incuba-
tion with bacterial DNA has been hotly contested (52, 53). In spite of
considerable effort in this area, including the direct uptake of exogenous
DNA by cell cultures, callus cells, and protoplasts (52, 66, 67, 68, 88)
there remains little convincing evidence that uptake of exogenously supplied
DNA offers potential usefulness for genetic transformation. An exception
is the recent report that plasmids isolated from a bacterium, Agrobacterium
tumefaciens, can directly transform Petunia protoplasts (20).
4.2.2. Transformation using biological vectors. There is some prom-
ising evidence which suggests that biological vectors may be utilized for
successful transfer of bacterial genes to plant cells. The most thoroughly
investigated vector is the crown gall bacterium, Agrobacterium tumefaciens.
It is well established that tumor-inducing strains of A. tumefaciens contain
large (100-150 megadalton) extrachromosomal, covalently closed, circular
DNA plasmids (76). The direct association of tumor-inducing properties of
A. tumefaciens with such plasmids (Ti plasmids) has been clearly demonstra-
ted. Strains of A. tumefaciens which have lost the Ti plasmid have also
lost the ability to induce tumors; introduction of a Ti plasmid into a non-
tumor-inducing acceptor strain by conjugation confers the capacity to induce
tumors in plant cells; deletion mutants of Ti plasmids are not able to in-
duce tumors (84). Recently it has been shown that a portion of the Ti
plasmid, termed T-DNA, is stably incorporated into chromosomal DNA of plant
tumor cells (17, 24, 95). Transformed plant cells produce a novel class of
amino acids referred to as opines. Naturally-occurring strains of A.
tumefaciens are unusual in that they can utilize opines as sole sources of
380

carbon and nitrogen. Interestingly, the genetic information for production

of the opines is contained on the Ti plasmid T-DNA. Therefore, the inter-
action of the pathogen and the host is an unusual type of parasitism, termed
genetic colonization (77), in which the pathogen injects genetic information
into the host cell and causes the host cell to produce metabolites that the
pathogen alone can utilize. Analysis of cultural, physiological and genetic
features of crown gall disease presents a unique opportunity for exploring
this naturally-occurring system of prokaryotic-eukaryotic genetic trans-
formation.
Applications are being made of this unique host-pathogen relationship,
as is elegantly illustrated by such studies as that of Matzke and Chilton
(60) in which a procedure is described for the insertion of specific genes
into the T-DNA. Use of the crown gall host-pathogen system may eventually
provide an avenue for genetic modification of angiosperm and some gymnosperm
forest species, since many are known to be susceptible to crown gall disease,
especially in the seedling stage. There are some difficulties, specifically
with regard to regeneration plants from transformed tumor and tumor-teratoma
cells. When such difficulties are overcome, the use of recombinant Ti
plasmids for production of genetically modified plants may become a reality.
Another vector offering potential for genetic modification of plants
is the plant DNA virus. Typical plant viruses contain RNA as the hereditary
molecule (e.g., tobacco mosaic virus). Some exist however, that contain DNA
genomes including caulimoviruses, geminiviruses and potato leaf roll viruses
(30). Of these, the cauliflower mosaic virus (CaMY) has been most inten-
sively characterized (45, 85). Several features of this potential trans-
formation vehicle may limit its us eful ness, however. CaMY has a limited
host range, and mainly infects members of the Brassicaceae. In addition,
the likely site of replication of CaMY is the host cytoplasm, rather than
the nucleus and ability of CaMY to interact with the nuclear genome of the
host cell may be limited. Although the use of DNA viruses to introduce
foreign genes into plant cells may be under some limitations, additional
studies examining the molecular events associated with basic host-virus
interactions may lead to new procedures for realizing this goal.
4.2.3. Pollen as a vector in genetic transformation. Work from Hess'
laboratory (36, 37, 38, 39, 40, 41) has centered on the uptake of phage
particles and bacterial, plasmid, and higher plant DNA by swelling and
germinating pollen grains. Modified pollen grains are then used as vectors
 381

for introduction of new genes into zygotes at the time of fertilization.

In the genus Nicotiana, sexual hybrids between N. langsdorfii and N. glauca
are characterized by the production of tumors. Using pollen of N. glauca
to take up DNA isolated from N. langsdorfii followed by self-pollination of
N. glauca with this pollen, Hess (41) has produced "transformants" that form
tumors and, thus, demonstrated possible use of this procedure in effecting
gene transfer. Additional work (37, 39) utilizing pollen transfer of bac-
terial genes for lactose and galactose utilization, however, have presented
somewhat unclear evidence of successful transfer and gene expression. In
spite of this, results are encouraging. Although pollen of some forest
species is difficult to germinate in vitro and may rapidly loose its via-
bility during storage, pollen transfer procedures may offer an additional
means whereby new genes may be introduced into plant cells. In many forest
trees, especially conifers, where regeneration of whole plants from modified
cells in culture presents problems, the pollen transfer procedure holds
particular promise, since the resulting transformed cells, zygotes, are
fully totipotent.

5. CONCLUSIONS
This review has presented a brief overview of recent sophisticated and
theoretical advances in genetic modification of plants using in vitro sys-
tems. It is readily apparent that very few reports have centered on appli-
cation to forest trees. Applications of such procedures, including in vitro
selection and somatic hybridization, await development of techniques for
production of plantlets from single cells or callus cultures of economically
important species. This must be our immediate research goal.
For the long term, it is felt that in vitro procedures will have appli-
cation in the development of genetically improved forest trees. Production
of intra- and interspecific hybrid lines will likely be achieved through
protoplast procedures. An understanding of fundamental aspects of naturally-
occurring genetic transformations, such as crown gall disease, may lead to
the harnessing of such systems for genetic improvement of trees. Simple
transformations may involve maintenance and expression of genes for single
peptides, such as those conferring characters as diverse as insect resistance
and attachment of nitrogen-fixing bacteria. Application is seen in terms
of expanding the ranges of certain forest species as well as increasing
yield.
382

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 387

13. VEGETATIVE PROPAGATION IN RELATION TO JUVENILITY, MATURITY, AND

REJUVENATION

J.M. Bonga

1. INTRODUCTION
For forest tree improvement it is essential to develop methods of vegeta-
tive propagation of mature trees, because the potential for obtaining better
forests through sexual breeding is limited. The major problem with sexual
breeding, besides the long life cycle of trees, is that many woody species,
especially those in temperate climate zones, are highly heterozygous (1, 64).
This limits the use of inbreeding and controlled hybridization as a means of
obtaining genetic gain because inbreeding of heterozygous plants often leads
to severe growth depression in the offspring (64, 93, 120) . Nevertheless,
with continuous breeding and selection over several generat i ons some tree
improvement can be expected (93), but the gain is likely to be less than if
selected trees are propagated vegetatively (Fig. 1). Another advantage of
mass vegetative propagation is that increased productivity is immediate, whereas
with sexual propagation it requires several generations. Furthermore, vegeta-
tive propagation allows mass propagation of hybrids and polyploids with low
sexual fertility (12).
Vegetative propagation of woody plants of a size large enough to have demon-
strated their potential is often difficult or impossible with the traditional
methods of rooting of cuttings and grafting. Cloning by means of tissue cul-
ture is an alternative to the traditional methods , and has been successfully
employed to obtain propagules from mature plants of some tree species (see
earlier chapters on vegetative propagation). However, most mature hardwoods
and conifers are still difficult or impossible to propagate by tissue culture.
In fact, tissue culture techniques often offer no advantage over traditional
rooting of cutting techniques because the capacity of a plant to propagate
vegetatively in tissue culture is often lost at an age when the plant can still
be propagated by rooting its cuttings (Fig. 2). For example, in many conifers
the capacity for embryogenesis and morphogenesis diminishes shortly after
388

germination (10), and in some species even before germination (75). There-
fore, to achieve vegetative propagation of hitherto "recalcitrant" plants,
some of the presently used tissue culture methods may have to be drastically
changed. To develop such new methods, factors such as juvenility, maturation,
determination in meristems, clonal deterioration, genetic stability, and
rejuvenation will have to be considered. Each of these factors must be
controlled before uniform, true-to-type, long-term cloning of trees can be
achieved.

Fig. 1. Comparison of genetic gains

obtained with vegetative and sexual
propagation of selected plus trees.
The upper curve shows the distribu-
tion of tree height in an even-aged
(f)
natural stand. There is considerablE
w variation in height. By selecting
W
0::
l-
the tallest trees (shaded area) and
LL sexually propagating these, the mean
o of heights is shifted slightly to
0::
w the right, i.e., there is a small
m
genetic gain; variability is somewhat
?5
z reduced (lower left curve). By vege-
tative propagation of the selected
trees the mean of tree heights is
shifted further to the right, Le.,
genetic gain is larger; variability
is reduced considerably (lower right
curve).

ISMALL'
17 LARGE .1

GENETIC GAIN

2. JUVENILITY - MATURITY
2.1. Definitions
The capacity to vegetatively propagate trees is associated with juve-
nility. Generally, the more juvenile the specimen, the easier it is to prop-
agate vegetatively. There is no clearly defined transition from the juvenile
to the mature phase in most plants. Often some parts of the tree may be
mature, or senescent, while other portions still display juvenile charac-
teristics. Furthermore, in some trees major morphological characteristics
 389

may change abruptly (heteroblastic) when maturing, while in others, the changes
are more gradual (homoblastic) (98).

*.!-~
1--1/!~~
I D
c\ 414-
~~
~1'i)/
E A

Fig. 2. The vegetative propagation cycle of many of the conifers . In vitro

propagation is generally only possible with sections of embryos (A) or young
germinants (B). This in vitro propagation capability is already lost in
young seedlings (C), but these can still be propagated by conventional root-
ing of cutting techniques. Cuttings taken from these seedlings root readily
and generally produce true-to-type propagules. Cuttings from a somewhat older
tree (D) have a reduced rooting capability, and the propagules often show
varying degrees of plagiotropic growth. Cuttings from mature trees (E) do
not root.

Taking these complications into consideration, the following terms will

be used in this chapter: 1) "Juvenile" are those plant parts or cells in
which there is a distinct preponderance of typical juvenile characteristics
over mature ones. For vegetative propagation purposes it is important to be
able to recognize which trees, or which tissues or parts of trees, are juve-
nile. Juvenile and mature characteristics are shown in Table I. This table
is an elaboration of an earlier one presented by Wareing (118). Juvenile areas
within the tree are shown in Fig. 3. Cells in tissue culture will be called
juvenile if they have the capacity for true-to-type vegetative propagation.
2) "Mature" are those plant parts or cells in which most or all of the juve-
nile characteristics have been replaced by mature ones. One prominent mature
390

Table I. Juvenile and Mature Characteristics a

Juvenile Mature

Nucleus Small b (8, 43, 77, 101)C Reticulated and

elongated (9)
Surrounded by a thin Surrounded by endoplasmic
cytoplasmic layer (8) reticulum membranes (8)

Chromatin Decondensed (124) Condensed (124)

Methylated DNA (50, 51)
Polyploid euchromatin
(77, 101)

Ribosomes Free (45) Membrane associated (45)

Apical meristems Small dome (43, 49, 100) High dome with increased
Large cell s (43) RNA (49)

Leaf Simple shape (82, 100) Complex shape (82, 100)

Large epidermal cells (100) Dense venation (82)
Retention in winter (100) Thick (82)

Branches Branch angle obtuse (.100) Branch angle acute (100)

Trunk Non-forking (100) Forking (100)

Short tracheids (98)
Smooth bark (100) Thick bark (88)

Cuttings Adventitious rooting easy Adventitious rooting

(100) often difficult (100)

Cells and tissues Capable of organogenesis Not capable of

in vitro organogenesis

a Some of the characteristics listed here are based on observations

made on only one or a few species or tissues, i.e., they may not apply to
all species or tissues.

b This obviously does not apply to the zygote, which has a large
nucleus.

c Reference number.
 391

Fig. 3. Diagram of juvenility gradients in trees. Left: Juvenility in a

regularly shaped conifer. The degree of juvenility of an apical meristem is
inversely proportional to the distance (along trunk and branches) between the
root-shoot junction (A) and the meristem. Since laterals are shorter than the
in ternodes they origina ted from, the dis tance AB >AC >AD >AE >AF, and therefore,
meristem B is the most mature, and meristem F is the most juvenile. Right:
Juvenility in several of the hardwoods. Density of crosshatching indicates
degree of juvenility. Epicormics(E), spheroblast(Sp), root sprouts(R), stump
sprouts(S), and severely pruned trees(P) are juvenile. In the juvenile zone,
note the single trunk, retention of leaves close to the trunk in winter, and
obtuse branch angles. In the mature zone, note the forked trunk and acute
branch angles.

characteristic of a tree is its capacity to flower. An important mature

characteristic of cells or tissues is that they have lost the capacity for
adventi tious organ or embryo formation. 3) "Partial rejuvenation" describes
the disappearance of at least some distinct mature characteristics and the
reappearance of some juvenile characteristics in plant parts, tissues, or
cells. Partial rejuvenation may go to the point where tissues or cells regain
the capacity of limited "morphogenesis", Le., of forming either adventitious
roots or shoots. 4) "Complete rejuvenation" occurs when mature or partially
matured tissues or cells regain the capacity of forming adventitious embryos
that will grow into normal plants. Sometimes adventitious embryos may arise
that remain stunted or are otherwise abnormal (14). Some of these may be the
392

result of environmental limitations, but others may have arisen from cells in
which some maturity was still retained, i.e., rejuvenation was almost, but not
entirely complete. 5) "Dedifferentiation" is often used in the tissue culture
literature to describe events similar to those described above under partial
and complete rejuvenation. However, in other literature the term dedifferenti-
ation often has other meanings than reprogramming of cells to a state in which
they are capable of adventitious organ or embryo formation. Therefore, the
term rejuvenation is preferred to describe increasing morphogenetic ability of
cells, but the term dedifferentiation may be used when discussing a literature
reference in which it is used.
2.2. Determination in meristems
Meristematic apices, the centers of growth and organization in plants,
undergo changes when the plant matures. Therefore, the tissues derived from
these apices behave differently in young and old plants (13, 43, 64, 100). One
consequence of this is that shoot cuttings from older trees often root poorly
or not at all. Furthermore, if rooting occurs, the propagules may not behave
true-to-type, but show undesired characteristics such as plagiotropic growth,
reduced growth rate, changed taper, etc. (51, 94). Differences between rooted
cuttings and seed-derived specimens are often maintained throughout the life of
the trees. For example, mature trees grown from rooted Pinus radiata cuttings
are different in several respects from trees grown to maturity from seed (65).
In some cases, specific growth charac teristics are very firmly "determined" 1
in the meristems. The classical example is Araucaria, where only cuttings fron:
vertical stems will form normal plants after rooting. Rooted cuttings from
primary and secondary branches, instead of growing vertically, have been ob-
served to maintain a horizontal growth habit for over 50 years, with the rooted
primary branch cuttings forming laterals, and with the rooted secondary branch
cuttings failing to do so (40, 44, 73, 100). Similar, but less extreme and
persistent trends have been observed in Larix, Picea, Pseudotsuga (40, 118) and
some hardwoods (40), while in Douglas fir plagiotropism is already noticeable
in rooted cuttings from seedlings only 5-7 months old (97).
It is obvious from these examples that true-to-type vegetative propagation
is often possible only with very young, juvenile material. However, as was

1 The term "determination" is generally used to indicate an epigenetically

controlled, relatively stable fixation of characteristics in cells and tissue
(73) Such determination in shoot meristems is often called "topophysis"
(84,100).
 J93

pointed out earlier, the objective is to find methods to propagate trees old
enough to have expressed their genetic potential. This indicates the need of a
close study of juvenility and of the possibility of experimentally obtaining at
least partial, but preferably complete, rejuvenation. Therefore, first it should
be determined which cells and tissues in the plant retain juvenility the long-
est, and use these tissues and cells as explants for in vitro propagation.
Next, those species which have propagated vegetatively naturally, sometimes over
thousands of years, should be investigated. How did these species maintain
their meristems in a sufficiently juvenile condition for true-to-type cloning
over many generations of propagules; how did they maintain their genetic
stability during those years? Lastly, it should be determined if partial or
complete rejuvenation occurs naturally, and if this process can be duplicated
experimentally.
Sometimes there are reasons other than vegetative propagation why main-
tenance or reestablishment of juvenility is of interest. The juvenile phase
of tree growth has some attributes which are important in a practical forestry
context. Firstly, when trees mature their growth rates often drop. Secondly,
in many hardwoods the trunk is single as long as the tree is juvenile, but
starts to fork when the tree reaches maturity (Fig. 3). Thirdly, juvenile
trunks have more radial growth, and juvenile bark is more resistant to dis-
ease and stress (84, 100, 112). However, juvenile characteristics are not
always the ones desired. For example, fibre length is short in juvenile wood
(33) and in conifers the mature growth form is sometimes better than the juve-
nile one (112, 113). Therefore, in some vegetative propagation programs cut-
tings of older trees are preferred over those of younger trees, because when
cuttings of older plants are rooted, the juvenile growth phase can be bypassed
resulting in straighter, less tapered stems, although, alas, with less volume
growth (112, 113).
2.3. Juvenile zones
Most trees have zones that retain a degree of juvenility longer than other
areas of the tree. For example, roots often retain juvenility and thus a capa-
city for vegetative propagation and juvenile type of growth, for a long time
(16, 82, 90, 117). In the above-ground part of the tree, the less distance a
shoot apical meristem is located from the base of the trunk, the more juvenile
it generally is. In a regularly shaped tree, like a conifer, this means that
the meristem at the apex of the leader is the least juvenile; the apical meri-
stem at the end of a first-order branch in the upper part of the tree is more
394

juveni1e; the apical bud of a first-order branch in the lower part of the tree
is more juvenile again, and the apical bud on a second order branch of this
lower branch is more juvenile still (in Fig. 3, AB>AC>AD>AE>AF) (61, 84). In
the tree, this zonation of juvenility is reflected in cuttings from lower
branches rooting easier than those from higher branches, and cuttings from a
higher branching order rooting easier than those from a lower branching order
(40) . Zonal differences in juvenility are also demonstrated by differences in
winter leaf retention, in apical meristem shape and cytochemistry (49, 100,
101), and in many other characteristics (Table I, Fig. 3). This increase in
juvenility towards the center and lower parts of the crown is exploited in
vegetative propagation practices. The most common practice is heavy and re-
peated pruning, or cutting down of the tree, thus forcing new shoot formation
from the lower and innermost branches or from the lower parts of the trunk
(Fig. 3). Most of these juvenile sprouts do not arise de novo from adventi-
tious buds, but from buds laid down early in the life cycle of the tree that
have remained strongly suppressed and thus juvenile (arrested juvenility) up to
the time of pruning or cutting (34, 39).
It has been suggested that the differences in degree of juvenility between
different shoot apical meristems in the tree are related to the number of cell
divisions that separate each meristem from the original embryo shoot apex. To
assume some loss in juvenility in each successive division (84, 95), in analogy
to what has been observed in lower organisms (78) and several animal cell cul-
tures (47, 109), is probably reasonable, but only up to a point, as will be
discussed later.
Differences in maturity in the above-ground parts of a tree are not con-
fined to apical meristems, but are evident also in the cambium. The cambium
near the base of the trunk is more juvenile than that in the upper part of
the trunk, and may retain some juvenile characteristics for many years (84).
This juvenility is expressed in the cambial derivatives by bark type, tracheid-
vessel ratio, and tracheid length (84, 88, 98).
2.4. Clonal aging
On the basis of the earlier discussed maintenance of mature characteris-
tics in rooted cuttings or grafts if the cuttings or scions are taken from
the mature part of the tree, one would expect that in successive generations
of cloning, including repeated in vitro cloning, mature characteristics would
accumulate progressively. In older literature it is often stated that repeated
vegetative propagation eventually leads to clone senility, especially in plants
 395

that in nature reproduce only sexually, and that this senility can only be
alleviated by going through a sexual cycle (40, 69, 88). However, this con-
cept of clonal degeneration is generally no longer accepted, most clonal de-
generation is currently ascribed to pathogen accumulation or adverse environ-
mental conditions (40, 46, 63). It appears therefore, that even though mature
characteristics may be transmitted to the first generation of propagules,
later generations do not progressively become more mature when propagated
regularly. In fact, many tree species, including those that predominantly
propagate sexually, have been cloned for hundreds or some even a few thousand
years without any signs of clonal degradation (28, 76, 116). Furthermore, in a
few clones instead of degradation, a degree of rejuvenation has been achieved
by application of special techniques. For example, a Vitis clone, which for
centuries has been propagated only in its mature form, reverted to the juvenile
form after several generations of in vitro repropagation of propagules when
these were still very small (76). Early repropagation, by rooting cuttings,
has resulted in arrested maturation in some species (70, 74), probably because
this procedure keeps the shoot apex close to roots (70, 82). However, the
opposite has also been observed. Robinson and Wareing (95) found that if black
currant was regularly cloned before reaching mature height, flower induction
became easier after a few generations of cloning, i.e., the clone had become
more mature. Clonal maturation and senility are distinct features in proto-
zoans, fungi, and other lower organisms. Growth slows down and can only be
restored to the original rate by a passage through the sexual cycle (42, 106).
Clonal senility also occurs in several animal cell cultures, but, in these,
unlike in most plant clones, senility is not caused by transmissible viruses or
other pathogens (109).
Many plant cell or tissue clones can be maintained in vitro well beyond the
normal life span of the plants that provided the cells or tissue for culture.
For example, callus cultures from tomato plants have maintained vigorous growth
continuously since 1945 (62), although it could be claimed that these cells are
transformed (habituated) cells, i.e., they are cells that are different from
the cells in the original explant and have lost the capacity to mature. Even
though seemingly immortal, long-term in vitro plant cell clones often deteri-
orate; chromosome numbers change and the competence for embryogenesis or orga-
nogenesis generally diminishes with subculturing (72).
396

2.5. Genetic stability

As was pointed out earlier, many tree species have propagated themselves
clonally for thousands of years. Presumably these species have not under-
gone much genetic change during those many years, which contrasts with the
rapid genetic changes found in many tissue culture propagation systems.
Genetic stability is not confined to clonal propagules, but is noticed
also in long-lived individuals in such species as Sequoia sempervirens and
Pinus aristata (up to 4000 years old, (69, which will form functional new
root and shoot primordia, free of genetic aberrations for many centuries.
Another indication of genetic stability in shoot meristems is that the cell
lines eventually entering meiosis have maintained the capacity to pass on to
the next generation, genetic information, which in spite of many years of
exposure to mutagenic environmental and physiological stresses, is still large-
ly unchanged. Similarly in very old trees, the vascular cambium, in spite
of prolonged environmental pressure, has retained its genetic and organizationa
integrity and thus the capacity to form functional phloem and xylem elements.
It is this stability of meristems that is being made use of when in vitro
propagation is achieved directly from the explants, without an intervening
callus or cell suspension phase. The disadvantages of this system are that
the rates of propagation are much lower than those potentially possible from
cell suspension cultures, and that genetic manipulation, selection for specific
traits, hybridization, etc. (Chapter 12), cannot be done as well as with cell
suspension cultures. It is important, therefore, to determine the mechanisms
of maintenance of regenerative capacity and of genetic stability in those
clones of trees that have propagated vegetatively for centuries without clonal
deterioraton, and, if possible, to incorporate these mechanisms in in vitro
systems of mass propagation of trees by callus or cell suspension cultures.
2.6. Mechanisms of maturation
As was pointed out earlier, the ability to propagate vegetatively diminish-
es with maturation. Therefore, maturation, at least in a few critical cells,
will have to be arrested and possibly reversed in any system of vegetative
propagation. To be able to counteract maturation, answers are required to
such questions as, where in the cell or tissue does it occur, and how?
In sexual propagation, the zygote is the first cell of each new organism.
It is a completely juvenile cell, i.e., it has the capacity to fully express
its genes 2 and form a new organism.
2 In some organisms cell determination begins before the egg is fertilized
(55), i.e., in some zygotes some repression is already present.
 3~

In the past, the prevailing view was that genes are gradually activated
during embryo initiation and later growth. Currently it is more generally
accepted that progressive gene repression dominates selective gene activa-
tion during development (24, 55). One mechanism of gene inactivation appears
to be methylation of chromosomal DNA (50, 51, 92); possibly others are euchro-
matization (101), or to the contrary heterochromatization and histone forma-
tion (52, 55, 107). However, maturation is not solely determined by the nucleus,
but also by the accumulation of self-replicating determinants in the cytoplasm,
which can be passed on through many cell generations (13, 15, 105, 107, 119).
These self-replicating determinants presumably are cytoplasmic organelles which
contain DNA and thus genetic information (4, 15, 42, 107), or they are free
cytoplasmic DNA released by disintegrating organelles (38). This organellar DNA
is circular and is structurally much simpler than chromosomal DNA, and although
sometimes present in large amounts, its genetic information is always limited in
kind (42, 57). However, organellar DNA may be a principal factor in determining
the level of maturation of a cell. For example, in fungi transfer of cytoplas-
mic DNA (probably of mitochondrial origin) from a mature mycelium into a juve-
nile one causes maturation of the latter (37, 38).
Even though maturation of a cell may, to a degree, be determined by the num-
ber and distribution of these DNA-containing organelles, or by DNA released by
these organelles into the cytoplasm, there are other factors that could be
important. For example, in time, organelles tend to differentiate structurally
to more complex forms (60,), which may affect the expression of their DNA, and
their DNA often becomes more polyploid or amplified (4, 38, 48).
The effects of the cytoplasm on the nucleus, and vice versa, during matura-
tion have been clearly shown in several nuclear transplant studies. For exam-
ple, in experiments with Acetabularia, mature cytoplasm induced rapid maturation
of immature nuclei (8, 15, 89). Conversely, if nuclei of mature cells were
implanted in the enucleated cytoplasm of young cells, the mature nuclei rejuve-
nated (8, 89). However, nuclear rejuvenation in a juvenile cytoplasm is not a
universal phenomenon; in nuclear transplant experiments with Paramecium, mature
nuclei did not rejuvenate in juvenile cytoplasm (17). Mature frogs have been
cloned by transplanting nuclei from skin cells into enucleated egg or embryo
cells (19, 24), although the success rate decreases as the maturation level of
the transplanted nucleus increases (19, 71). Therefore, nuclear rejuvenation is
possible, but only if maturation has not progressed too far. Conceivably, in
many mature plants and animals only those somatic cells that will eventually
398

enter meiosis may have nuclei at a maturation level low enough to be rejuvena-
ted by juvenile cytoplasm.
2.7. Mechanisms of juvenility retention
From earlier comments (section 2.3.) one would be inclined to conclude
that the degree of maturity of a cell or meristem of a tree is proportional
to the number of cell divisions that separate the cell or meristem from the
zygote. With respect to the general principle of maturation, this statement
is probably correct, but with regard to different cells in different loca-
tions within the plant or meristem it is an oversimplification, because: 1)
In some cell lines, the cells may mature more in each division than those in
other cell lines. 2) If maturity of a cell is partly determined by cyto-
plasmic determinants, unequal divisions would result in differences in matur-
ity of the two daughter cells because of unequal distribution of the cyto-
plasm between the two cells (13). 3) In some cell lines, a mechanism may be
present for occasional or regular, partial rejuvenation of the cells (see
later).
Nevertheless, limiting the number of cell divisions probably favors juve-
nility retention. On that basis, the following mechanism is suggested to
explain retention of juvenility in some tissues, especially in long-lived
clones. In all major plant meristems there are tissues with a low mitotic
activity, and the function of this low mitotic activity may well be to main-
tain a pool of relatively juvenile cells within the meristems. In the more
active areas of the root and shoot apical meristems, the cells divide and
organize lateral root, shoot, or leaf primordia. In this process, the cells
presumably become slightly more mature in each cell division, and eventually
reach a level of maturity where cell division is still possible but the
capacity to form primordia is lost. When this stage is reached, some cells
may be removed from the juvenile low activity portions of the meristem to
replenish the active tissue with relatively juvenile cells (3, lOS, 114).
The low activity zones are not completely inert; their cells are dividing
occasionally at a low rate, and thus one would expect a slow gradual matura-
tion even in these low activity areas. However, to explain how very long-
lived trees such as sequoia and bristlecone pine (69), and long-lived clones
such as poplar (2S) and creosote bush (116), manage to produce functional new
shoot and root primordia after centuries of active growth, we probably have to
assume, that a counteracting, rejuvenating mechanism is present in at least a
few cells in the low activity areas. Following this concept further, species
 399

that are difficult to propagate vegetatively probably differ from easily prop-
agated species in that the maturation - rejuvenation balance in the low activ-
ity zones in the latter is such that the cells are maintained at a more juve-
nile level.
Relatively inactive zones are found in root tips, shoot tips, and the cam-
bium. Of these, the quiescent center in the roots is generally the least
active . It possesses great regenerative capacity; if the root apex is damag-
ed, a new root will arise from surviving quiescent cells (3, 114). In the
vegeta ti ve shoot apex, the central zone, sometimes called the "meris tern d' a t-
tente", is relatively inactive (6B, 96, lOB) as long as the shoot remains veg-
etative. In developing flowers the apices behave differently. Flowers are
"determinate" structures (lOB), I.e., they die after they have performed their
flowering function. Therefore, there is no need to maintain a pool of rela-
tively inactive cells in the central zone of the flower apex, and the division
rate in this zone increases (21, lOB), as some of the cells are prepared for
meiosis.
The central zone of the shoot apex has been traced back to a few specific
cells of the embryo, and remains relatively inactive as it is carried along
inside the shoot apex from the time of germination to the onset of flowering
(110). Thus, the central zone in the apex of a tall, old tree has undergone
only a limited number of divisions since its conception in the germinating
embryo.
Another major meristem with a relatively inactive zone is the cambium,
where the rate of division is much lower in the cambial initials than in adja-
cent cells (lOB, Ill). Long-term retention of function in the cambium also
may depend on the relative inactivity of some of its cells, i.e., of the cam-
bial initials.
2.B. Mechanisms of genetic stability
Genetic stability is a prerequisite for successful cloning, especially if
the cloning is repeated over several successive generations. Therefore, it is
important to discover how this stability is maintained in meristems of long-
lived trees and natural clones. Again, low mitotic activity may be crucial,
because gene mutation rates are proportional to the rate of division (23), and
therefore, good genetic stability can be expected in relatively inactive areas.
Evidence of genetic stability of inactive areas in meristems has been pro-
vided by experiments with root tips. The quiescent center of the root tip is
very stable in adverse environmental conditions. For example, it is relatively
400

insensitive to damage caused by X-rays, y-, or a-irradiation, mutagenic chemi-

cals like 5-aminouracil, cold shock, or other treatments that affect nuclear
integrity (26, 27, 96, 114).
A mechanism that is perhaps involved in maintaining genetic stability in
the low activity areas of meristems is non-random segregation of DNA strands
during division of specific cells. This system was first discovered in ani-
mals (23, 91), and has recently been found in plants (56, 115). The principle
characteristics of the system are: 1) The specific cells remain in a more or
less fixed position within an organ. 2) When they divide, the daughter cell
containing the original DNA strand remains in the original position in the
organ in a relatively inactive state; the other daughter cell, i.e., the one
containing the new DNA strand, will go through several rapid divisions thus
providing the bulk of the tissue or organ. 3) The cell with the original DNA
strand is temporarily activated each time the cell line started by the daugh-
ter cell with the new DNA has gone through so many divisions that it is start-
ing to degenerate functionally. 4) Most DNA errors arise during replication.
Therefore, keeping the original, undamaged DNA in reserve in a few cells in a
strategic location within the organ, creates a genetically stable system. In
short, this system is effective in maintaining both organ function and genetic
stability over long periods of time.
2.9. Mechanisms of rejuvenation
Rejuvenation of cells and tissues is probably the single, most important
aspect in achieving effective cloning. As was discussed earlier (section
2.6.), maturation of the nucleus can sometimes be reversed by transplanting
the mature nucleus to a juvenile cytoplasm. The question now arises, what is
the nature of the rejuvenating signals transmitted by the juvenile cytoplasm
to the nucleus? There is evidence that some rejuvenating signals, "morphogen-
etic substances", are mRNA's (15, 89). In other references, it is claimed
that the organelle to nucleus (and vice versa) messengers are primarily pro-
teins (42, 71).
With trees, little work has been done along these lines. Nuclear trans-
plants, as a means of cloning, have never been reported, but initial attempts
to associate RNA and low molecular weight proteins with morphogenesis have
been made, using in vitro cultures of Douglas fir cotyledons (123).
Whenever shoots arise adventitiously, in plant tissue culture or in situ,
they are juvenile (43, 69). However, as was pointed out earlier, in cultures
of tissues of mature trees such rejuvenation is achieved only with a few
 401

species. This warrants a closer look at the one stage in the life cycle of
every tree where complete rejuvenation always occurs, namely during sexual
reproduction. If we could duplicate this meiotic rejuvenation mechanism in
somatic cells without reducing the number of chromosomes, asexual propagation
from somatic tissues of mature trees would likely become possible.
2.10. Sexual rejuvenation
Since the subject of sexual rejuvenation has been reviewed earlier (13),
the subsequent discussion will be confined mostly to literature not considered
at that time.
It has been claimed, that some cellular dedifferentiation occurs in the
prefloral (21), and premeiotic (82) stages. However, the most dramatic reju-
venation probably occurs during the meiotic prophase (13). During the meiotic
prophase, there is a drastic reduction in numbers of cytoplasmic ribosomes and
plastids, and a simplification (dedifferentiation) of mitochondria (30, 67,
102). The apparent function of this process is to remove residual, long-lived,
ribosome-associated mRNA from the cytoplasm during the transition from the
mature sporophyte to gametophyte and male and female gametes (30, 31), and
results, after fertilization, in a completely juvenile zygote. The reduction
in complexity of organelles, or their partial removal, is accomplished by lys-
ing enzymes, primarily acid phosphatase and ribonuclease (30). The most like-
ly sources of these enzymes are vacuoles, cell walls, and membranes (86, 87),
although the lysosomal function of the vacuole has been questioned (53).
Lysing during meiosis is not restricted to the cytoplasm. A somewhat simi-
lar process occurs within the nucleus when vacuoles and other structural com-
ponents of the sporophyte nucleus are reconditioned to those of a gametophyte
nucleus (103). Whether the genetic information of the nucleus is reprogrammed
at the same time for the juvenile phase of growth is not known; it could be
reprogrammed later, either shortly before, during, or even after fertilization
(50).
Plastid and ribosome destruction is not total during the meiotic prophase.
A small number of these organelles is sequestered from the rest by membranes
and thus is partially protected from lysing (30). Initially, the sequestered
organelles undergo little degradation and presumably remain sufficiently func-
tional to properly maintain cell metabolism while the rest of the cytoplasm is
devoid of functional organelles. During the later phases of meiosis, the
cytoplasm is rapidly repopulated with new (presumably juvenile) organelles.
402

New mitochondria are formed from the partially lysed, structurally simplified
ones (67, 102), and new ribosomes are released by the nucleus (30, 102).
It is not clear what happens to organellar DNA during meiosis. Earlier
(section 2.6.) it was pointed out that DNA in the cytoplasmic organelles prob-
ably is directly involved in part of the maturation process in the cell.
Therefore, it is important to establish what happens to the cytoplasmic DNA
during meiosis. Does it survive during organelle dedifferentiation or elimi-
nation, what happens to its ploidy, and does it become reprogrammed during
meiosis? These are just a few of the many still unanswered questions.
It has been found that tumor-inducing plasmid DNA disappears from the cyto-
plasm during meiosis (18, 122). At present, it is not known if the DNA from
the regular organelles is similarly eliminated, but if so, where would the new
DNA for new organelles come from? During meiosis there is an abundance of
circular extrachromosomal DNA in the nucleolus organizer in the nucleus of
some organisms (52). It is not known if this is a source for new cytoplasmic
organellar DNA. Observations suggesting release of de novo organelles with
extra-chromosomal DNA from the nucleus have been described (6, 7), but have
also been questioned (4, 32).
In conclusion, it appears that old information is being removed from the
cytoplasm during meiosis. However, it is not yet clear how the cytoplasm is
subsequently provided with new information, and how and when the nuclear
genetic information is reprogrammed. A better understanding of the mechanisms
of cytoplasm and nucleus rejuvenation during meiosis is important because it
may suggest means of removing the mature determination from cells of mature
trees, thus reestablishing their capacity for somatic embryogenesis and true-
to-type vegetative propagation.
3. SIGNIFICANCE FOR PROPAGATION BY TISSUE CULTURE
If we are faced with a recalcitrant in vitro culture from which true-to-
type vegetative propagation does not materialize, the following general ap-
proach should be considered.
It probably is wise to first develop methods for vegetative propagation
from highly juvenile material, i.e., from sections of embryos or very young
seedlings. Once propagation from juvenile material has been achieved, and the
basic environmental parameters for culture of the species have been establish-
ed, the following procedures are suggested for mature material. 1) It may be
necessary to select explants containing cells that will eventually enter meio-
sis, because these may be the only cells in the plant body in which the nuclei
 403

have not matured past the point where reprogramming of the nucleus for embryo-
genesis has become impossible. However, it is not currently known how far the
nuclei in non-meiotic tissues have matured, and it is possible that the nuclei
of at least some tissues other than those entering meiosis could be completely
rejuvenated for use in somatic embryogenesis. 2) Since genetic stability and
juvenility are probably best retained in cell lines that are separated from
the embryo by low numbers of mitosis (section 2.7.), it is recommended that
tissues containing such relatively inactive cell lines be cultured. It is
also advisable to limit cell division between tissue excision and induction of
morphogenesis, i.e., an intervening callus stage should be avoided as much as
possible. 3) Based on the discussion on rejuvenation during meiosis, it is
recommended that tissues be selected for culture which have recently undergone
a reduction in number or complexity of cytoplasmic organelles.
Obviously, the choice of explant is important, and therefore, a survey is
presented below of which parts of the tree, excluding the embryo, would be
most suitable for excision and culture.
3.1. Choice of explants
3.1.1. Flower parts. Somatic tissues of flowers of many plants have a high
capacity for vegetative reproduction, possibly because of their proximity to
the rejuvenating sexual cells (81, 82, 83). With regard to trees, morphogene-
sis has been obtained in cultures of somatic tissues of sexual shoots of sev-
eral species (12, 14, 35). According to some authors (21, 82), a dedifferent-
iation of cells occurs just before or shortly after flower induction. There-
fore, explants should probably be taken from flowers at an early developmental
stage. One part of later stage flowers that is of considerable interest is
the nucellus. In some citrus varieties, adventitious embryos arise naturally
from the nucellus (20, 58, 61), but the propagules raised from these do not
always develop true-to-type (58). Adventitious embryos have also been obtain-
ed in vitro from nucellar tissues of citrus (22) and apple (36). In view of
earlier comments regarding rejuvenation of the cytoplasm during meiosis, it is
of interest to note that during development of the nucellus the cytoplasmic
organelles in its cells are reduced in number and become disorganized (79, 80).
3.1.2. Vegetative buds. Vegetative buds, or parts thereof, have frequently
been used as explants in experiments designed to obtain vegetative propagation
of trees (see chapters on vegetative propagation). The central part of the
apex is probably the most responsive. Its cells have low mitotic rates and
404

low numbers of ribosomes (21), both of which, as was pointed out earlier, may
be significant in relation to the morphogenetic capacity of the tissue.
In cultures of shoots dissected from buds of mature conifers, small, shoot-
like structures were obtained, mostly at the base of young needles (14). Many
of these structures may have arisen from the lining of the resin ducts in the
needles, which again is a tissue with structurally simple plastids and, in the
later stages of its development, a low number of ribosomes (25, 121).
3.1.3. Roots. Shoot buds will form naturally near the apex of roots of
some species and have been induced in root cultures of others (90). They will
form only rarely from the root apical meristem itself, i.e., the root apical
meristem, like the shoot apical meristem, is firmly determined, and the two
meristems normally are not interconvertible.
3.1.4. Root-shoot junction. This area may contain arrested juvenile buds
(section 2.3.) which will flush into juvenile sprouts if the tree is cut down
or severely pruned. In vitro culture of such juvenile sprouts has resulted in
clonal propagation of, for example, sequoia (2). However, in many tree spec-
ies such sprouts are not formed, and thus are not available for cloning pur-
poses.
As was emphasized throughout this section on explants, many of the tissues
suitable for vegetative propagation have low numbers of organelles, or struc-
turally simple organelles. Of course, one may not automatically assume that
this condition is always causally related to morphogenesis. For example, a
reduction in numbers and complexity of organelles is a common feature in sen-
escing tissues (5, 38). Senescence generally does not favor morphogenesis,
although both are not always mutually exclusive. For example, in citrus cul-
tures, aging of the callus resulted in increased embryogenesis (54).
3.2. Chemical and physical methods of reducing organelles
It is still far from clear, whether the mechanisms of reprogramming of
cells for morphogenesis are indeed similar to the mechanisms of rejuvenation
during sexual reproduction. Nevertheless, it may be worthwhile to determine
if some of the processes occurring during sexual rejuvenation can be duplicat-
ed in somatic cells by chemical or physical means, and if this is subsequently
followed by morphogenesis. Since the first step, and possibly the most impor-
tant one, in sexual rejuvenation appears to be a reduction in number and com-
plexity of organelles, possibly accompanied by a removal of organellar DNA,
this process should receive some attention. An alternative approach would be
 405

to partially inhibit organelle function, or inhibit its DNA, without destroy-

ing or simplifying the organelles.
Since lysing of organelles is mainly achieved by hydrolytic enzymes releas-
ed from vacuoles, cell walls, and membranes, presumably any treatment that
will increase the release of these enzymes to the cytoplasm could be effect-
ive. Many stress-inducing treatments would fall in that category, and the
process may not be very specific (5). 'Perhaps some of the morphogenetic ef-
fects of many chemicals not normally considered growth regulators, for example
NaOH and H2S0 4 which stimulate rooting (59), or such treatments as starva-
tion (6, 54), coldshock, or centrifugation (99) could in part be explained by
stress-associated partial destruction of organelles and their DNA.
Of the many chemicals that structurally reduce organelles (42), only the
terpenes (essential oils) will be mentioned, because they remove organelles
naturally (25, 66). They are produced in abundance in the resin duct lining
of expanding needles, resulting in a reduction in the number of organelles in
these cells (11, 25). As was noted earlier, in mature conifers these cells
are among the few that are capable of limited morphogenesis. Other indica-
tions of a possible relationship between terpenes and morphogenesis are that
terpene-containing secretory cells dedifferentiate easier than parenchymatous
and other cells (41), and that sesquiterpenes stimulate adventitious root for-
mation (104).
An alternative to partial destruction of organelles and their DNA could be
a temporary partial blocking of the organellar DNA functions, possibly by an-
tibiotics, or ethidium bromide (4, 42). Ethidium bromide inhibits aging in
fungi (37, 38), and some antibiotics stimulate shoot initiation (85).
At this point it should be stressed that although partial destruction or
simplification of organelles, and possibly removal of their DNA, may have a
function in rejuvenation during meiosis, the process probably involves many
other essential changes. Therefore, until more is known about meiotic rejuve-
nation, it remains an open question if simply removing organelles or their DNA
from somatic cells will stimulate morphogenesis.

4. SUMMARY AND CONCLUSION

Within the tree there are tissues in which juvenility, and genetic stabil-
ity are better maintained than in other tissues. To obtain true-to-type vege-
tative propagation, it is important to select the most juvenile tissues and
406

insure that morphogenesis is induced in these as quickly as possible, i.e., a

long callus phase should be avoided.
Somatic rejuvenation occurs naturally or is experimentally induced in some
tissues in some mature trees, and may progress to a point where these tissues
become capable of organogenesis or embryogenesis. However, in mature trees of
many species such natural or induced rejuvenation does not occur or does not
go far enough to support morphogenesIs. To achieve, by experimental means,
somatic rejuvenation in these "recalcitrant" species is still a major chal-
lenge.
Maturation occurs in both the nucleus and cytoplasm. Since maturation is
probably easier to reverse in the cytoplasm than in the nucleus, and because a
rejuvenated cytoplasm may automatically rejuvenate the nucleus if maturation
of the latter has not progressed too far, the development of methods to repro-
gram the cytoplasm should receive attention first. A complete rejuvenation of
the cytoplasm occurs during meiosis. One known event in this rejuvenation is
a reduction in number and complexity of cytoplasmic organelles. Whether a
similar condition could be experimentally induced in the cytoplasm of somatic
cells, and whether this would result in rejuvenation of these cells, remains
to be assessed.
All mechanisms involved in the complete rejuvenation occurring during sex-
ual reproduction should be studied. Attempting to duplicate these mechanisms
in cultures of somatic cells of mature trees may well be the most promising
approach to eventually achieve true-to-type propagation of these trees.

5. ACKNOWLEDGEMENTS
I wish to thank Dr. W.K. Coleman, Canada Department of Agriculture, and Dr.
J.E.A. Seabrook, University of New Brunswick, Fredericton, Canada, for review-
ing the manuscript.

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6. BELL PR 1963 The cytochemical and ultrastructural peculiarities of the

fern egg. J Linn Soc (Bot) 58: 353-359
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 413

14. TREE SPECIES INDEX

Abies, 317 Coffea, 23

balsamea, 21,23,77,88,89,105,317, arabica, 126,127,146
318 canephora, 146
nordmanniana, 73 Cornus, 110
sacha1inensis, 244,246 Cory1 us
Acac ia, 132,133 ave11ana, 128,143,146
koa, 121,143,237,242 Cryptomer ia
Acer, 110,145 japonica, 83,107,234,242,246
pseudop1atanus, 14,237,256,281, Cupressus, 315,331
320,321,322,323,324,328,329, arizonica, 80,108,242,375
330,331,333,334,335,336,341, funebris, 244,330
345,348,356 macrocar pa, 242
rubrum, 112,143,147 sempervirens, 242
saccharum, 326,331,335 Cycas
Aesculus, 110 circinal is, 73, 105
hippocastanum, 143
Ail an thus , 110
Albizia, 46 Dalberg ia, 46,110
Alnus, 23,46
glutinosa, 131 Elaeis
rubra, 131,143 guineensis, 183,188,190,194
Artocarpus Ephedra
heterophy11us, 237,238 gerardiana, 74,105,319,330
Araucar ia Eucalyptus, 16,46,52,65,110,112,150
cunninghamii, 392 alba, 143,156,234,236,237
astr ingens, 155
Banbinia, 10 bancroftii, 155,156,157,160,237,
Baphia, 46 238
Betula, 110,132 bicostata, 152
1 utea, 39 botyroides, 152
pendu1a, 121,143,234,237,240,370 bridgesiana, 158
platyphylla, 125,143 camaldu1ensis, 152,153,154,155,
schezuanica, 125 156,157,158,165,166,168,237
Biota citriodora, 156,235,243
orientalis, 74,84,85,88,108,240, cl adocal yx, 152,156
242,313 curtissii, 158
Bomb ax, 110 dalrymp1eana, 151,157,158,169
Boswell ia, 110 deg1upta, 152,153,154,157,162,163,
Broussonetia, 111 166
kazinoki, 143 delegatensis, 151,155,163,169
fastigata, 152
Cal iandra, 46 ficifo1ia, 154,157,158,165,166,
Carya, 22 170,240,241,242,244
il1inoensis,143 fraxinoides, 155
Castanea, 110,222,322 gigantea, 155
sativa, 143,234,237,240,241 globulus, 158
vesca, 243 gomphocephala, 156,158
Casur ina, 111 grandis, 143,152,153,154,155,156,
Catalpa 157,158,159,160,161,162,163,
bignonioides, 143 164,165,166,167,168,236,237,
Chamaedorea, 189 324
costaricana, 185,190,193,199,200 gunnii, 151,156,157,158,169,172,
seifrizii, 185 173,174
Citrus, 17,21,403 1aevopinea, 155,156
Cinnamornum, 111 macarthur ii, 157
Cocos macul ata, 156
nucifera, 183,185,190 me11 iodora, 155,156,157,163
414

nichollii, 155,156,160 Liquidambar, 110,133,146,148,301

nitens, 155 styraciflua, 119,124,128,143,291
nova-angl ica, 156 Liriodendron, 110
obl iqua, 156,163 tulipifera, 124,125,143
obtusifolia, 158
pauciflora, 151,157,158,169 Malus, 21,22,213,326,327,403
platyphylla, 154 Michel ia, 111
pol ybractea, 156,158,166 Morus, 110
regnans, 155,158
robusta, 143,152,156 Nyssa, 110
saligna,152,156
tereticor nis, 155,156 Paulownia
trabuti, 156 taiwaniana, 143
transcontinetal is, 154 tomentosa, 122,128,143,146
urnigera, 155,156,157,158 Phoenix
urophylla, 156 atl antaca, 187
v iminal is, 156 dactylifera, 183,184,187,190,192,
193,200
Fagus recl inata, 187
silvatica, 124,143 roebel enii, 185
Ficus, 111 picea, 22,24,313,373,392
Fraxinus, 17,110,327 abies, 73,74,81,84,85,86,89,91,93,
excel sior, 350 94,105,234,235,237,242,243,245,
pensyl vanica, 354 266,280,314,317,318
excel sia, 237
glauca, 39,52,84,88,89,98,105,
Garuga, 111 237,238,240,241,242,243,245,
Ginkgo 270,272,288,293,296,313,314,
biloba, 24,88,279,290,313,314, 315,316,317,318
315,316 mariana, 39,106,317
Gleditsia sitchensis, 74,81,93,106,240,242,
triocanthos, 143 243
Gmel ina, 46,111 Pinus, 22,46,65,274,313
albicaulis, 237
Hevea aristata, 396,398
brasil iensis, 143 banksiana, 25,39,84,106,237,240,
Howeia 262,279,293,317,318
fosteriana, 185,189,190,193,198 cembra, 237
Hymenodictyon, 111 clausa, 313,314,315
contorta, 74,80,81,93,106,240,
Ilex 242,243
aquifo1 ium, 127,146 coulterei, 17,265
echinata, 39,242
Jugl ands, 110 elliottii, 242,245,314,315,320
Juniperus, 331 fl ex il is, 237
communis, 315,316,319 gerardiana, 73,106,236,237,238,
317
Keteleer ia halepensis, 319
davidiana, 234 jeffreyi, 315
lambertiana, 234,317,351
Lagerstroemia, 110,163 monticola, 223,238,245
Lannea, 111 palustris, 80,91,98,106,242,314,
Larrea tridentata, 398 315,316
Larix, 317,392 pinaster, 77,78,80,81,82,83,85,
eurolepis, 79,105 90,92,93,96,97,98,99,106,170,
occidental is, 237 236,237,242,245,316,317,375
Leucaena, 46 pinea, 319
Libocedrus ponderosa, 242
decurrens, 330 radiata, 20,74,75,81,84,91,93,95,
,98,106,236,241,242,243,339,
353,354,356,378,392
 415

resinosa, 39,88,106,290 Rhapis, 189

rigida, 242 excelsa, 185
roxburghii, 234,244,246 Robinia, 145
sabiniana, 242 pseudoacacia, 119,122,144,147
scrotina, 313,314
strobus, 39,76,82,94,107,223,235, Sabal
237,242,243, 245,315 pal metto, 197
sy1vestris, 82,83,85,94,98,106, Sal ix, 109,110
234,235,240,241,242,245,246, baby1onica, 144
291,317 Santal um
taeda, 17,58,61,75,107,215,218, album, 126,144,146,234,236,237,
222,240,242,243,256,281,293, 238,240,242,243,244
314,319, Sequoia
virginiana, 242 gigantea, 80,107
wa11ichiana, 75,82,89,107,242 sempervirens, 12,72,79,80,90,92,
yunnanensis, 234 94,96,97,107,158,276,313,315,
Platanus, 22, 110 316,396,398,404
occ idental is, 112 Shorea, 111
Populus, 22,23,109,110,121,132,145,
212,321,322,373,398
alba, 123,124,144,242 Taxus
anescens, 242 baccata, 244,246,316
canadensis, 144 Tectona, 23,110,124
canescens, 242 grandis, 144,234,237,238,242,243,
del toides, 123 244
euroamericana, 120,124,144,242 Terminalia,lll
glandu1osa, 242 Theobroma
nigra, 123,124,144,236,237,240, cacao, 127,146
241,242,243 Thuja, 317
popul us, 144 occidental is, 242
robusta, 245 p1icata, 80,84,98,108,234,236,
simonii, 237 241,242,244
tremula, 123,124,144,234,241,242
Toona, 110
tremuloides, 115,116,123,144,236, Tsuga
237,238,240,241,242,243 heterophylla, 81,98,107,234,237,
ussuriensis, 237,240 242
yunnanensis, 123
Prosopsis, 46 Ulmus, 110,373
Prunus, 23, 110 americana, 121,144,288,377
persica, 112 campestris, 115,121,124,144, 242,
serotina, 39, 125 320
Pseudophoenix effusa, 124,144
sargent ii, 197 scabra, 124,144
Pseudotsuga
menzies~~, 24,50,55,65,74,75,76, vitis
77,80,81,85,88,89,91,92,93,94, vinifera, 216,395
96,98,107,223,234,235,236,237,
238,240,242,243,245,247,256, Washingtonia
270,274,277,280,281,287,293, filifera, 197
317,318,319,353,372,374,376, robusta, 197
392,400
taxifolia, 107,238,245 zamia
Pterocarpus, 46 floridana, 80,105
integrifolia, 73,88,105,316
Quercus, 110
robur, 124,144
416
15. GENERAL INDEX
abscisic acid, 22,73,163,246,259
agar, 16
concentration, 16
impurities, 16
substitutes, 16,372,376
age,
reduced morphogenesis, 87,109,166
air layering, 152,186
amino acids, 256
conjugates, 263,297
relation to growth regulators, 261
systhesis, 258,260
ammonium, 17,89,258,269,294
anther culture, 157,295,375
antibiotics, 332,370,405
ascorbic acid, 196
antioxidant, 24,198
unstable, 20
autoclaving, 10
chemical decomposition, 11,90
auxins, 231
autoclaving effects, 12
conjugates, 233,298
effect of phenolics, 299
morphogenesis, 72,232,297
pH, 298
polar transport, 215,245,298
bacteria, 212
benzylaminopurine, 36,241
morphogenesis, 37,73,115,241
nitrogen metabolism, 257
stability, 12,241
brachyblasts, 77,79,85,93,96
buds (also see shoots),
adventitious, 80
ammonium effect, 82
axillary, 77,89,116,122,123,154
170,191,199
carbohydrates effect, 339
induction correlated to parent
condition, 20
inhibitors, 163
on roots, 117,118
origin, 82
sucrose effect, 352
suppressed (arrested), 111,154,394
buffers, 18
cambium,
culture, 21,72,115,121,244,370
inactive zone, 325
carbohydrates, 325
decomposition in autoclaving, 11
embryogenesis, 337
metabolism, 325, 329
nutri tion, 326
uptake, 328,334
cells,
amino acid composition, 271
carbohydrate metabolism, 329,334
embryogenesis, 126,136,293
internal divisions, 295
mitotic,
phases, 265,284,334
rates, 394
nitrogen metabolism, 267,280
organogenesis, 55,57,120
suspensions, 13,53,73,174,372
ultrastructure, 287,299
walls, 285,299,325,330,341
cellulose, 46,345
charcoal, 17,89,171,198,277
impurities, 18,277
ion exchange, 18
stimulates morphogenesis, 17,90,96
chemicals,
impurities, 278
instability, 7,19,25
sterilization, 12
storage, 7
chromatin, 264,290,297,390
climate,
cycles, 20
effect on culture, 20
cotyledon culture, 77,86,116,122,127,
192,244,247,293,296,339,356,374,400
culture vessels, 19
cytokinin, 239
autoclaving effects, 12
effect on membranes, 239
morphogenesis, 232,239,297
nitrogen metabolism, 261
rejuvenation, 170
dedifferentiation, 295,392
determination, 388,392
2,4-dichlorophenoxyacetic acid, 73,232
chromosomal changes, 238
disease,
control, 38,53,98,202,208
infection mechanisms, 217
inhibitors, 218
resistance, 220,370,373

DNA,
breaks, 25
cell cycle, 265
cytoplasmic, 402
gene expression, 264,301,381,396
methylation, 397
non random segregation, 400
uptake, 379
EDTA, 18,272
chelate, 18
forcing, 24
inhibits ethylene formation, 18
stimulates,
embryogenesis, 18
nitrate reductase, 18
organogenesis, 18,24
embryo,
culture, 72,80,122,156,190,195,
244
nutrition (natural), 270,289
pelleting (fluid drilling), 53,136
embryogenesis, 88,126,136,146,156,
176,189,201,291,293,296,372
abnormal, 391
carbohydrate metabolism, 17,337
juvenility, 387
nitrogen metabolism, 272,280,291
epicormic branches, 111,154,391
ethylene, 15,18,24,246,261
nitrogen metabolism, 261
Eucalyptus, 150
cold hardiness, 169
industrial aspects, 168
vegetative propagation,
tissue culture, 152
traditional, 155
excision,
antioxidants, 23,198
phenolics, 23,159,198
transfer hoods, 6,7
explants,
between tree variation, 131
response depends on,
season, 21,85
seed size, 20
exudates, 159,191,284
fermentors, 14,59,280,334
flavonoids, 25,161,299
fructose, 11,88,326
fungi, 216
galactose,
morphogenesis, 17
417
nutrition, 326,332
toxic, 17,332
genetics, 264,369,387
aneuploids, 120,192
cold resistance, 151,157,168,169,
370
engineering, 56,369,378
gene expression, 264,301,381,396
haploids, 269,279,369,375,377
heterozygosity, 186
inbreeding, 186,387
natural variation, 370
polyploids, 120,387,397
selection, 36,369,371
stability, 52,87,120,155,192,201,
277,369,396,399
gibberellic acid, 78,88,90,165,171,
195,231,244
autoclaving effect, 12
carbohydrate metabolism, 334
cell growth, 231,244
embryogenesis, 244
nitrogen metabolism, 261
organogenesis, 245,339
synthesis, 259
glassware,
sterilization, 10
storage, 7
vessels, 19
glucose, 88,326
glutamine,
autoclaving effect, 12
dehydrogenase, 257
effect of auxin, 298
intracellular, 282
protoplast culture, 376
synthesis, 260
unstable stored, 19,20
GOGAT enzyme, 257
grafting, 96,132,152,378
growth rooms, 14
mites, 15
pre-conditioning of plant
material, 20
volatiles, 15
gymnosperms, 72
cell suspension culture, 53,73,
267,280,293
embryogenesis, 55,76,293
gene action, 266
genetic stability, 52
industrial aspects, 36
organ culture, 52,77
organogenesis, 52,55,73,294,296
regeneration from mature plants, 96
418

hardwood (dicot) trees, 109,150 seed orchards, 49

cell suspension culture, 126,281
291
industrial aspects, 36,113,129
vegetative propagation,
tissue culture, 114,155
traditional, 109,152
heteroblastic, 151,389
histones, 264
hybrids, 56,151,189,202,374
hypocotyl culture, 52,77,83,116,122,
133, 272, 291, 296
species specific requirements, 131
tissue culture, 37,41,50,129
tropical hardwoods, 48
vegetative propagation, 49,109,
118,150
inflorescense,
culture, 191,199,403
reverting to vegetative shoots,
186,188,200,202
insects, 215
2-isopentenyl purine, 73

incubation, juvenility, 52,86,96,100,109,111,116,

fermentors, 13 126,151,158,268,296,387,390
shakers, 13
physical factors, 17,19,24 kinetin, 74,88,240
indoleacetic acid, 231
cell growth, 231 laboratory,
conjugates, 233,298 dishwashing, 6
detection, 234 facilities for,
morphogenesis, 37,74,234 excision, 6
nitrogen metabolism, 257 incubation, 13
oxidase, 233,248,298 transfer, 6
pH, 233 layout, 5
unstable, 20,233 sterilization, 10
indolebutyric acid, 231,235 storage of,
morphogenesis, 37,73,112 chemicals, 7
stability, 235 glassware, 7
indolepropionic acid, 238 water purification, 8
industrial aspects, 36 lectins, 60,281,300,343
biochemical transformation, 59 light,
cell suspension culture, 53 dark pretreatment, 25
cellulose, 46 decomposition of chemicals, 25
costs, 64,113,129,135,147,148, intensity, 14
175 mutagenic, 25
disease control, 38,64,202,208 near ultraviolet, 25
energy, 45,59 quality, 24,296
fiber, 47,136,393 lignin, 345
genetic, lignotuber, 155,156
resources, 44
selection, 36,38,41,111,131, mannitol,
151,168,196,209,301,373 nutrient, 17,327
variability, 131 osmoticum, 17,353,375
greenhouse requirements, 113,129 maturation, 396
gymnosperms, 36 meristems,
hardwood (dicot) trees, 46,48,113, d'attente, 399
168 determination, 392
hybrids, 56,151 maturation, 393,398
leguminous trees, 46 mitotic rates, 398
multiple use, 46,64 mites, 15
palms, 182 mitochondria, 22,340,355
pel1eting (liquid drilling) of mutagens, 25,371,400
embryos, 53,136 mutants, 60,63,269,369
productivity, 38,40,111,126,145,199 myo-inositol, 296,344

mycorhiza, 94,95,98
naphthaleneacetic acid, 36,235
morphogenesis, 37,112
stability, 235
nematodes, 214
nitrogen, 256
fixation, 46,61,62,212,300,378
levels in tissue, 276
metabolism, 256,301,336
natural compounds, 257
nitrate/reduced nitrogen, 291
reductase, 18,258
node culture, 157,163
nucellus, 403
nucleic acids, 41,259,297
effect of carbohydrates, 335
nitrogen metabolism, 261
systhesis, 284
nutrient media, 15
buffers, 18
carbohydrates, 326
charcoal, 17,18,89,171,198,277
growth regulators, 231
minerals, 16,79,93,95,170,290,295
ni trogen, 256
osmoticums, 17,349,350,353,375,378
pre-conditioning, 127,283
storage, 19
oils, 183
organ culture, 77,122,157,168
organelles, 264,286,328,377,397,401,
404
organogenesis, 52,55,73,294,296,372
auxin/cytokinin, 246
carbohydrate metabolism, 337,352
cell walls, 286,299
gene action, 264
gymnosperms, 52,55,73,294,296
hardwood (dicot) trees, 116,117,
156
juvenility, 387
on needles, 84
palms, 190,201
phenolics, 246,299
proteins, 82
seasonal effect, 21,85
ultrastructure, 287,299
osmoticums, 17,349,350,353,375,378
ovary culture, 157,193
oxidases, 95,215
palms, 182
carbohydrates, 184
4.19
genetic stability, 192,201
heterozygosity, 186,188
inflorescence reverts to
vegetative shoot, 186,188
oils, 183
ornamental, 185
vegetative propagation,
tissue culture, 190
traditional, 185
pectins, 342
pentose phosphate pathway, 283,333,
336,347,348
pentoses, 326,331,335
phenols, 23,49,90,95,159,161,198,262,
298
phloroglucinol, 162
phytoalexins, 209
phytotoxins, 209,213
plagiotropism,97,152,389,392
plant material,
collection, 20
storage, 20
varies with season, 21,85,154
plasmids, 212,213,379,402
polyamines, 268
polyethylene gas exchange, 15
polyphenoloxidase, 160
polyvinylpyrrolidone, 23,49,160
potassium,
embryogenesis, 17
nitrogen metabolism, 261
organogenesis, 78,79
potting mixtures, 95,120,124,125,132,
169,173
pre-conditioning (plant material),
chemical treatments, 86
climatic factors, 20,163
cold treatment, 162
effect on nitrogen metabolism, 275
in field, 53
in greenhouse, 20
protein,
chromosomal, 265
glyco, 343
nonhistone chromosomal, 264
organogenesis, 297,400
systhesis, 259
protoplasts, 56,100,210,268,329,344,
369,374
pruning, 112,131,155,164,394
rejuvenation, 96,153,391,395,397,400
respiration, 260,282,333
inhibitors, 328,329
rhizobia, 62,378
420

root, transfer to soil, 25,98,120,123,125,

amino acids, 95 132,173
buds, 117,118,404 triiodobenzoic acid, 245
cultures, 157 trueness to type, 52,63,99,116,120,
formation, 91,95,112,166,296 121,388,392,403
inhibitors, 154,162,191
juvenility, 393 urea, 88,261,269,273,282
origin, 191
quiescent zone, 399 vascular development, 295,327,338,
sucrose, 351 347,396
precocious, 295
sexual, vegetative propagation,
propagation, 396 carbohydrate metabolism, 325
rejuvenation, 395,401 clonal deterioration, 388,394
shakers, 14 conventional methods, 109,152,185,
shoot (also see buds), 387
apices, 116 Eucalyptus, 150
elongation, 88,171 genetic gain, 388
tip culture, 135,148,211,403 gymnosperms, 36,72,256,387
sodium hypochlorite, 23,199 hardwood (dicot) trees, 36,109,
sphaeroblasts, 56,288,293,391 150,256,387
starch nutrition, 326,331 juvenility, 387
sterilization, 10 mature trees, 64,96,115,122,124,
"Agarmatic", 11 131,154,198,277,387
autoc1aving, 10 nitrogen metabolism, 256
chemical decomposition, 11 palms, 182
chemicals, 10 sexual versus vegetative, 387
dry, 10 viruses, 210,380,395
filter, 12 vitamins instability, 12,25
glassware, 10
with, water, 8
alcohol, 12 deionization, 9
dimethylsulfoxide, 12 distillation, 8
purity, 8
storage, reverse osmosis, 9
chemicals, 7 storage, 10
cryopreservation, 22,200
nutrient media, 19 zeatin, 241,243
plant material, 20,174
stump sprouts, 79,110,111,169,276,391
sucrose,
autoclaving effects, 11
embryogenesis, 351
exudates, 160
nitrogen metabolism, 260,283
nutrient, 327,330
osmoticum, 17,349
rooting, 351
vascular development, 327
surface sterilization, 22
methods, 22,199
problems, 22,157,158

tannins, 161
terpines, 405